Use of MIF and MIF Pathway Agonists

ABSTRACT

The present invention relates to novel methods and compositions for increasing AMPK activity and glucose uptake comprising administering a macrophage migration inhibitory factor (MIF) pathway agonist in a subject in need thereof. The invention also relates to methods for selecting a subject for treatment with an agonist of MIF, identifying a subject at risk for developing a condition in which increased AMPK activity is desirable, and for predicting whether a subject is susceptible to a condition in which increased AMPK activity is desirable.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/083,131, filed Apr. 3, 2008, which is a U.S. national stage filingunder 35 U.S.C. §371 of International Application No. PCT/US2006/039315,filed on Oct. 6, 2006, which claims priority under 35 U.S.C. §119(e)from U.S. Provisional Patent Application No. 60/725,146, filed Oct. 7,2005 and U.S. Provisional Patent Application No. 61/740,422, filed Nov.29, 2005, all of which are herein incorporated by reference in theirentirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

The invention described herein was supported, in whole or in part, bythe National Institute of Health Grant Nos. R01 HL63811, and AI4320X.The United States government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Glucose uptake by cells is a critical process for maintaining cellularenergy levels and ultimately, for tissue function and integrity.Increased glucose uptake has been shown to be cytoprotective in a numberof settings. Increasing cellular glucose uptake also is advantageous insome metabolic conditions characterized by impaired glucose transportand in organs and tissues that have high energy requirements. Anunderstanding of the mechanisms and pathways involved in cellularglucose uptake, thus, may permit the development of means for modulatingthe uptake process.

An important regulator of both glycolysis and glucose uptake in striatedmuscle is the AMP-activated protein kinase (AMPK). AMPK is aserine-threonine protein kinase that senses the cellular energy state,effecting multiple metabolic and non-metabolic pathways to increasecellular ATP production while also limiting energy consuming pathways(Young et al., Trends Cardiovasc Med 15, 110 (2005)). AMPK is regulatedallosterically via AMP binding to its gamma regulatory subunit and byphosphorylation of Thr172 in the activating domain of the catalyticalpha subunit by upstream kinases, including LKB1 and CaMKKβ (Baron etal, Circ Res (2005)).

AMPK is activated under conditions that lead to inadequate blood flowand associated tissue ischemia, such as coronary artery disease. In theheart, AMPK directly stimulates PFK-2 activity and glycolysis (Marsin etal., Curr Biol 10, 1247 (2000)), induces GLUT4 translocation andincreases ischemic glucose uptake preserves high energy phosphatecontent and limits myocardial injury and apoptosis (Russell, 3rd et al.,J Clin Invest 114, 495 (2004)). AMPK also stimulates skeletal muscleglucose transport (Bergeron et al AJP 1999) through a mechanism that isindependent of the insulin signaling pathway; thus AMPK provides atarget for the therapy of people with type 2 diabetes in whom theinsulin stimulated pathway of glucose transport is impaired, leading tohyperglycemia. Since vascular disease, including coronary arterydisease, peripheral arterial disease and cerebrovacular disease arehighly prevalent in type 2 diabetes, AMPK represents a highly desirabletarget for both the treatment of the metabolic derangements of type 2diabetes and the prevention of ischemic injury in these patients.

Accordingly, there is a critical need for methods and compositions forpreventing cellular and tissue injury resulting from tissue ischemia. Inparticular, there is a need for methods and compositions for increasingcellular glucose uptake and preventing the depletion of energy storesduring energetic stress associated with ischemia utilizing the AMPKpathway.

SUMMARY OF THE INVENTION

The present invention provides novel methods for increasing AMPKactivity and glucose uptake comprising administering MIF or a MIFpathway agonist (a “MIF agonist”) in a subject in need thereof.

In one embodiment, the invention provides a method of selecting asubject for treatment with a MIF agonist, wherein the subject has, or isat risk of developing, a condition in which increased AMPK activity isdesirable, comprising genotyping the subject for the presence of apolymorphism associated with decreased MIF expression, wherein a subjecthaving a polymorphism associated with decreased MIF expression isselected for treatment with a MIF agonist.

In another embodiment, the invention provides a method of identifying asubject at risk of developing a condition in which increased AMPKactivity is desirable, comprising genotyping the subject for thepresence of a polymorphism associated with decreased MIF expression,wherein a subject having a polymorphism associated decreased MIFexpression is at an increased risk of developing a condition in whichincreased AMPK activity is desirable.

In another embodiment, the invention provides a method of predicting theseverity of a condition in which increased AMPK activity is desirable ina subject, comprising genotyping the subject for the presence of apolymorphism associated with decreased MIF expression, wherein a subjecthaving a polymorphism associated with decreased MIF expression is at anincreased risk of developing a more severe condition in which increasedAMPK activity is desirable.

In other embodiments, the invention provides a method of predictingwhether a subject is susceptible to a condition in which increased AMPKactivity is desirable, comprising genotyping a subject for the presenceof a polymorphism associated with decreased MIF expression, wherein asubject having a polymorphism associated with decreased MIF expressionis more susceptible to the condition.

Polymorphisms associated with decreased MIF expression include: thepresence of five, or fewer than five, CATT repeats in the −794 region ofone or both alleles of the MIF gene or the presence of guanine atposition −173 of one or both alleles of the MIF gene. In one embodiment,a polymorphism associated with decreased MIF expression is the presenceof five CATT repeats in the −794 region of both alleles of the MIF gene.In another embodiment, a polymorphism associated with decreased MIFexpression is the presence of five CATT repeats in the −794 region ofboth alleles of the MIF gene and the presence of guanine at position−173 of one or both alleles of the MIF gene.

Polymorphisms associated with increased MIF expression include: thepresence of six or more CATT repeats in the −794 region of both allelesof the MIF gene or the presence of a non-guanine nucleotide at position−173 of both alleles of the MIF gene.

In certain embodiments, a method of genotyping a subject for thepresence of a polymorphism associated with decreased MIF expressioncomprises contacting a sample obtained from the subject with apolynucleotide probe that hybridizes specifically to a sequencecomprising a polymorphism associated with decreased MIF expressiondetermining whether hybridization occurs. Hybridization indicateswhether the subject comprises a polymorphism associated with decreasedMIF expression, thereby genotyping the subject for the presence of apolymorphism associated with decreased MIF expression. In otherembodiments, the method further comprises contacting the sample with acontrol polynucleotide probe, wherein the control polynucleotide probedoes not hybridize specifically to a sequence comprising a polymorphismassociated with decreased MIF expression. Hybridization of thepolynucleotide probe but not the control polynucleotide probe indicatesthe presence of a MIF polymorphism associated with decreased MIFexpression.

In other embodiments, a method of genotyping a subject for the presenceof a polymorphism associated with decreased MIF expression comprises:(a) contacting a sample obtained from the subject with a pair ofamplification primers, wherein the primers are capable of amplifying aportion of the MIF promoter comprising a polymorphism associated withdecreased MIF expression; (b) amplifying DNA in the sample, therebyproducing amplified DNA; and, (c) determining whether the amplified DNAcomprises a polymorphism associated with decreased MIF expression,thereby genotyping the subject for the presence of a polymorphismassociated with decreased MIF expression. In certain embodiments, themethod comprises sequencing the amplified DNA.

In another embodiment, the invention provides a method of treating orpreventing a condition in which increased AMPK activity is desirable,comprising administering to a subject in need thereof a MIF agonist. Insome embodiments, a MIF agonist is administered to a subject having apolymorphism associated with decreased MIF expression. In otherembodiments, a subject having a polymorphism associated with decreasedMIF expression is administered a greater dose or amount of a MIF agonistthan a patient having a polymorphism associated with high MIFexpression.

In some embodiments, a method of treating or preventing a condition inwhich increased AMPK activity is desirable further comprises atherapeutic regimen that includes one or more additional treatmentmodalities.

In another embodiment, the invention provides a method of increasingphosphorylation of threonine at position 172 of the AMPK protein in acell, comprising administering a MIF agonist to a subject in needthereof.

In another embodiment, the invention provides a method of increasingAMPK-mediated GLUT4 activation in a cell, comprising administering a MIFagonist to a subject in need thereof.

In another embodiment, the invention provides a method of increasinguptake of AMPK-mediated glucose into a cell, comprising administering aMIF agonist to a subject in need thereof.

In another embodiment, the invention provides a method of increasingAMPK-mediated glycogen synthesis in a cell, comprising administering aMIF agonist to a subject in need thereof.

In yet another embodiment, the invention provides a method ofstimulating AMPK-mediated PFK-2 activity in a cell, comprisingadministering a MIF agonist to a subject in need thereof.

In other embodiments, the invention provides a method of increasingAMPK-mediated glycolysis in a cell, comprising administering a MIFagonist to a subject in need thereof.

In some embodiments, the invention provides a composition comprising oneor more MIF agonists and at least one AMPK agonist. In otherembodiments, the invention provides a composition comprising one or moreMIF agonists and at least one additional therapeutic agent.

Additional therapeutic agents may include, for example, compounds fortreating a subject having, or at risk of developing, ischemia orconditions related to ischemia, organ transplant surgery, or conditionsin which AMPK-mediated glucose uptake is desirable. In a specificembodiment, a condition in which increased AMPK activity is desirable istype 2 diabetes.

Conditions in which increased AMPK activity is desirable include, forexample, hypoxia, especially hypoxia resulting from tissue ischemia. Theischemia may be from any cause including acute coronary syndromes suchas myocardial infarction, coronary revascularization (such as coronarybypass surgery and coronary angioplasty/stent placement), stroke, renal,retinal, mesenteric or limb ischemia due to vascular occlusion, organtransplant surgery (for maintaining viability and function of thetransplanted organ), ischemia associated with vascular surgery,including hypothermic arrest and vascular cross-clamping. Otherconditions in which increased AMPK activity is desirable include, forexample, conditions in which AMPK-mediated glucose uptake is desirable.In a specific embodiment, a condition in which increased AMPK activityis desirable is type 2 diabetes.

A MIF agonist may be, for example, a MIF polypeptide, a CD74 agonist, aCD44 agonist, a bivalent antibody that increases the interaction betweenMIF, CD74 and CD44, a bivalent antibody that increases the interactionbetween MIF and CD74, or a bivalent antibody that increases theinteraction between CD74 and CD44. A MIF agonist may also include apolynucleotide or cDNA molecule that encodes any of the above proteins,including a MIF polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H are graphs showing MIF-associated AMPK signaling in isolatedpapillary muscles. FIG. 1A: Top. AMPK alpha subunit (α1 or α2)isoform-specific kinase activity from rat heart left ventricularpapillary muscles incubated in either oxygenated (100% O₂) or hypoxic(100% N₂) buffer for 30 minutes (n=3 per group, *P<0.01 vs. isoformcontrol and between hypoxic α1 and α2). Bottom. Representativeanti-phospho Thr172 AMPK immunoblots from papillary muscle homogenates.FIG. 1B: Papillary muscle MIF production; incubation buffer was sampledevery 5 minutes via ELISA during either control (oxygenated) or hypoxic(30 minutes) incubation (n=3 per group, P=0.03 ANOVA). FIGS. 1C and 1D:Anti-phospho Thr172 AMPK immunoblot densitometry, representativeimmunoblots, and papillary muscle 2-deoxy-D-[1-³H]glucose uptake frompapillary muscles incubated in hypoxic (100% N₂) buffer for 30 minutes,with or without pre-incubation with anti-MIF antibody or non-immune IgG(both 100 ug/ml) (n=3 per group, *P<0.05 vs. control, #P<0.05 vs.hypoxia alone). FIG. 1E: Anti-phospho Thr172 AMPK representativeimmunoblots and immunoblot densitometry from normoxic papillary musclesincubated with rMIF (0-400 ng/ml) for 60 minutes (n=3-4/condition,*P<0.02 vs. control). FIG. 1F: Anti-phospho Thr172 AMPK immunoblotdensitometry from normoxic papillary muscles incubated with rMIF (400ng/ml) for 0-120 minutes (n=3-4/condition, *P<0.05 vs. control). FIG.1G: Papillary muscle 2-deoxy-D-[1-³H]glucose uptake during incubationwith rMIF (0-800 ng/ml) for 60 minutes (n=6-8 per group, overallP<0.0001, *P<0.05 vs. control. FIG. 1H: Cell-surface GLUT4representative immunoblots and immunoblot densitometry measured usingthe cell-impermeant exofacial photolabel, bio-LC-ATB-BGPA, afterpapillary muscle incubation with rMIF (400 ng/ml) for 60 minutes (n=4,*P<0.001).

FIGS. 2A and 2B show cardiomyocyte MIF localization and ischemic cardiacMIF release. FIG. 2A: (top) Representative anti-MIF immunohistochemistry(40×) from formalin-fixed, paraffin-embedded sections of WT and MIF−/−hearts following control perfusion. Primary antibody either anti-MIF ornon-specific rabbit IgG developed with DAB and counterstained withhematoxylin. (bottom) Whole-heart lysates from WT and MIF−/− heartsimmunoblotted for MIF. Total AMPK was used as a loading control. FIG.2B: (left) Coronary effluent [MIF] measured by ELISA from WT heartsduring both control perfusion or ischemia/reperfusion. No MIF wasdetected in MIF−/− hearts' coronary effluent (n=3, *P=0.01). (right)Whole-heart lysates from WT hearts immunoblotted for MIF after controlperfusion or ischemia/reperfusion (n=3, *P=0.003).

FIGS. 3A-3C show ischemic AMPK activation, glucose uptake, andpost-ischemic cardiac function in isolated perfused MIF−/− hearts. FIG.3A: Anti-phospho Thr172 AMPK immunoblot densitometry, representativeimmunoblots. (left) and isoform-specific kinase activity. (right) fromtissue lysates from MIF−/− or WT hearts subjected to either controlperfusion or ischemia/reperfusion (immunoblots: n=4 per group, *P<0.05WT ischemia vs. MIF−/− ischemia, #P<0.05 ischemia vs. control; kinaseactivity n=3 per group). (B) Cardiac glucose uptake determined from theproduction of tritiated water from [2⁻³H]glucose (n=5 for each genotype,*P=0.01 vs. WT basal, #P=0.04 vs. MIF−/− reperfusion). (C) Cardiac ratepressure product (heart rate×left ventricular developed pressure) duringbaseline perfusion period and after 15 minutes of global, no-flowischemia (n=12 for each genotype during basal perfusion, 5 for eachgenotype during post-ischemic reperfusion, P=0.03 by repeated measuresANOVA during reperfusion).

FIGS. 4A and 4B show AMPK activation correlation with MIF promotergenotype in hypoxic human fibroblasts. FIG. 4A: Fibroblast incubationmedia [MIF] measured by ELISA from human fibroblasts subjected to eithercontrol incubation (95% room air; 5% CO₂) or hypoxia (95% N₂; 5% CO₂)for 9 hours, expressed relative to cellular lysate protein concentration(*P=0.03 vs. 5/5 control, #P=0.05 vs 5/5 9-hours of hypoxia, &P=0.03 vs.5/5 control). Data combined from fibroblasts with MIF promoter −794CATT_(5-E) tetranucleotide repeat polymorphisms of both 5-CATT repeatalleles (‘5/5’ genotype) or any combination of 6, 7, or 8-CATT repeatalleles (‘non-5/5’ genotype) (5/5 n=3, non-5/5 n=4). FIG. 4B:Anti-phospho Thr172 AMPK immunoblots from human fibroblasts subjected tocontrol or hypoxic incubation, with or without the addition of 10 ng/mlrMIF. (expressed relative to total AMPK, experiments run in triplicate,*P=0.01 control vs. 9-hours of hypoxia, #P=0.04 5/5 vs. non-5/5 9-hoursof hypoxia).

FIGS. 5A-5D show the characterization of the cardiac phenotype of MIF−/− mice. FIG. 5A: Heart weight (wet) and body weight measured fromage-matched WT and MIF −/− mice at 12-20 weeks of age (n=13/group). FIG.5B: Transthoracic 2-dimensional echocardiographic images of the leftventricle were obtained in the short axis using a 15 MHz probe on aPhillips Sonos 5500 machine. Mice were lightly anesthetized with inhaledisoflurane. Measurements were made in triplicate by a blinded observer(n=4 per group). FIG. 5C: Representative hematoxylin/eosin stainedsections of formalin-fixed, paraffin-embedded hearts (n=2-4 per group).FIG. 5D: Immunoblots of total AMPK alpha, CaMKKβ, LKB1, ACC, GLUT4,GLU1, and MIF from WT and MIF −/− hearts (n=3-4 per group).

FIG. 6 shows cardiac glycogen content in WT and MIF −/− hearts. (top)Cardiac glycogen content measured using the amyloglucosidase methodafter KOH digestion after control perfusion (baseline), 15 minutes ofischemia, or 15 minutes of ischemia/30 minutes of reperfusion (n=3-4 pergroup, *P<0.03 versus baseline). (bottom) Calculated ischemic glycogenbreakdown (baseline minus ischemia) and reperfusion glycogen synthesis(reperfusion minus ischemia) from group means above.

FIG. 7 shows loft ventricular systolic and diastolic pressure (top) and+dp/dt (bottom) in isolated WT and MIF−/− hearts during perfusion in theLangendorff mode. Left ventricular balloon volume initially set toachieve a diastolic pressure of 5 mmHg during baseline perfusion(n=10-12 per genotype, P=0.0001 WT vs. MIF−/− during reperfusion,ANOVA).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel methods for increasing AMPKactivity and glucose uptake comprising administering MIF or a MIFpathway agonist (a “MIF agonist”) in a subject in need thereof.

The methods and compositions of the present invention are useful totreat or prevent conditions in which AMPK-mediated glucose uptake isdesirable. Such conditions include hypoxia, especially hypoxia resultingfrom tissue ischemia. The ischemia may be from any cause including acutecoronary syndromes such as myocardial infarction, coronaryrevascularization (such as coronary bypass surgery and coronaryangioplasty/stent placement), stroke, renal, retinal, mesenteric or limbischemia due to vascular occlusion, organ transplant surgery (formaintaining viability and function of the transplanted organ), ischemiaassociated with vascular surgery, including hypothermic arrest andvascular cross-clamping.

Where tissue ischemia is anticipated to occur in a subject, such as asubject about to undergo a surgical procedure involving the interruptionor the substantial reduction of blood flow to a tissue or organ, orwhere the subject is at risk for an ischemic event, the methods andcompositions of the present invention also are useful to pretreat thesubject with one or more MIF agonists to increase AMPK activation andglucose uptake prior to the onset of anticipated hypoxic or ischemicinsult. For example, prior to coronary revascularization, such ascoronary bypass surgery and coronary angioplasty/stent placement. Theadministration of one or more MIF agonists also is advantageous prior tovascular surgery including hypothermic arrest and vascularcross-clamping. In addition, patients with symptoms or syndromesindicating imminent risk for severe ischemia may benefit from theadministration of MIF agonists. For example, patients with unstableangina, who are at risk for heart attack or those with transientischemic attack, who are at risk for stroke.

The methods and compositions of the invention also are useful inpatients suffering from a metabolic condition in which it isadvantageous to increase cellular glucose uptake, for example, inpatients with type II diabetes.

Subjects who may benefit from increased AMPK activity, e.g., to preventcellular and tissue injury due to tissue ischemia, hypoxia, or otherrelated conditions, or to increase glucose uptake, include but are notlimited to subjects who carry common polymorphisms in their MIF genesthat are associated with reduced MIF expression.

It is a further discovery of the present invention that differential MIFexpression governed by such polymorphisms has consequences in stresssignaling, specifically reducing hypoxic AMPK signaling that can be“rescued” by the administration of MIF.

Patients with low-MIF expression polymorphisms in whom it would beadvantageous to increase AMPK signaling and cellular glucose uptake,thus, may particularly benefit from treatment with a MIF agonist.Accordingly, the invention provides methods for identifying candidatesfor treatment with one or more MIF agonists to increase AMPK activationand for predicting therapeutic response to such treatment by genotypingthem to detect the presence of a polymorphism associated with reducedMIF expression.

It will be understood by one of ordinary skill in the art that thecompositions and methods described herein may be adapted and modified asis appropriate for the application being addressed and that thecompositions and methods described herein may be employed in othersuitable applications, and that such other additions and modificationswill not depart from the scope hereof.

A used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “including” is used to mean “including but not limited to”.“Including” and “including but not limited to” are used interchangeably.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule (such as a nucleicacid, an antibody, a protein or portion thereof, e.g., a peptide), or anextract made from biological materials such as bacteria, plants, fungi,or animal (particularly mammalian) cells or tissues. The activity ofsuch agents may render it suitable as a “therapeutic agent” which is abiologically, physiologically, or pharmacologically active substance (orsubstances) that acts locally or systemically in a subject. Agents cancomprise, for example, drugs, metabolites, intermediates, cofactors,transition state analogs, ions, metals, toxins and natural and syntheticpolymers (e.g., proteins, peptides, nucleic acids, polysaccharides,glycoproteins, hormones, receptors and cell surfaces such as cell wallsand cell membranes. Agents may also comprise alcohols, alkyl halides,amines, amides, esters, aldehydes, ethers and other classes of organicagents.

A “patient”, “subject”, or “individual” are used interchangeably andrefer to either a human or a non-human animal. These terms includemammals, such as humans, primates, livestock animals (including bovines,porcines, etc.), companion animals (e.g., canines, felines, etc.) androdents (e.g., mice and rats).

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may befurther modified, such as by conjugation with a labeling component. Theterm “recombinant” polynucleotide means a polynucleotide of genomic,cDNA, semisynthetic, or synthetic origin which either does not occur innature or is linked to another polynucleotide in a nonnaturalarrangement.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration of a drug and/or a non-drug modality (such as,for example, surgery, radiation, electrotherapy, diet, nutrition orexercise) to a patient. If it is administered prior to clinicalmanifestation of the unwanted condition (e.g., disease or other unwantedstate of the host animal) then the treatment is prophylactic, i.e., itprotects the host against developing the unwanted condition, whereas ifadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorateor maintain the existing unwanted condition or side effects therefrom).

As used herein, the term “MIF” or “MIF polypeptide” refers to macrophagemigration inhibitory factor or active fragments thereof. Accessionnumber EMBL Z23063 describes the nucleic acid sequence encoding humanMIF (Bernhagen et al, Biochemistry 33:14144-14155 (1994)). An activefragment of MIF may comprise a fragment or a portion of the MIF proteinencoding the tautomerase enzymatic activity of MIF, or a fragment thatis capable of binding CD74.

As used herein a “MIF agonist” refers to any agent that mimics,activates, stimulates, potentiates or increases the biological activityof MIF. A MIF agonist may be MIF or a fragment thereof; an agent thatmimics MIF (such as a small molecule); an agent that increases orenhances the expression of MIF, CD74 or CD44; an agent that enhances thebinding of MIF to CD74; an agent than enhances the interaction betweenCD74 and CD44 (including, without limitation, a bivalent agent).

As used herein, the “biological function of MIF” refers to the abilityof MIF to carry out one or more of the biological functions of KBincluding, without limitation, sustaining immune cell survival oractivation, promoting cytokine promotion, down-regulating CCR5, bindingto CD74, activating AMPK signaling, activating MAP kinase signaling(e.g., ERK1/2 JNK, and SAPK MAP kinase signaling), inhibiting p53,acting as a tautomerase, and/or acting as a thiol reductase.

As used herein, the term “treating” refers to preventing, slowing,delaying, stopping or reversing the progression of a condition.

As used herein, “a polymorphism associated with altered MIF expression”refers to any polymorphisms in the MIF gene that correlate withincreased or expression of the MIF gene, including without limitation: asingle nucleotide polymorphism (G/C) at position −173 of the MIFpromoter or the presence of five, six, seven or eight GATT repeats atposition in the −794 region of the MIF promoter.

As used herein, “a polymorphism associated with reduced MIF expression”refers to the presence of a guanine (G) at position −173 of one or bothalleles of the MD gene or the presence of five, or fewer than five, CATTboxes at position in the −794 region of one or both alleles of the MIFgene. (The positions of the MIF promoter are defined by reference to thenucleic acid sequence disclosed in EMBL Z23063.)

As used herein, “a polymorphism associated with increased MIFexpression” refers to the presence of a cytosine (C) at position −173 ofboth alleles of the MIF gene or the presence of six or more CATT boxesat position in the −794 region of both alleles of the MIF gene. (Thepositions of the MIF promoter are defined by reference to the nucleicacid sequence disclosed in EMBL Z23063.)

Each subject has two alleles corresponding to the MIF gene. As usedherein, “a subject having a polymorphism associated with reduced MIFexpression” refers to a subject that has a G at position −173 of the MIFgene in one or both alleles or that has five, or fewer than five, CATTrepeats in the −794 region of the MIF gene in one or both alleles. Asused herein, “a subject having a polymorphism associated with increasedMIF expression” refers to a subject that has a polymorphism associatedwith increased MIF expression in both of alleles of the MIF gene.

As used herein “higher risk” or “increased risk” refers to astatistically higher frequency of occurrence of the disease orcondition. As used herein “lower risk” or “decreased risk” refers to astatistically lower frequency of occurrence of the disease or condition.

As used herein, the term “severity” of a condition, refers to theseriousness, degree or state of a disease or condition. For example, acondition may be characterized as mild, moderate or severe. A person ofskill in the art would be able to determine or assess the severity of aparticular condition. For example, the severity of a condition may bedetermined by comparing the likelihood or length of survival of asubject having a condition with the likelihood or length of survival inother subjects having the same condition. In another embodiment, theseverity of a condition may be determined by comparing the symptoms ofthe condition in a subject having a condition with the severity of thesymptoms in other subjects having the same condition.

As used herein, the term “therapeutically effective amount” refers tothe amount of a MIF agonist (isolated or recombinantly produced), or acomposition comprising a MIF agonist, that is in sufficient quantitiesto increase AMPK activity, increase glucose uptake and/or delay, reduceor prevent cellular tissue injury from hypoxia, including from tissueischemia. For example, an effective amount is sufficient to delay, slow,or prevent the onset or progression of a condition associated withimpaired insulin-mediated glucose uptake such as in type 2 diabetes.

As used herein, the terms “isolated” and “purified,” when used inrelation to a nucleic acid, protein or other compound, refer to theseparation of the nucleic acid, protein or other compound from at leastone contaminant with which it is ordinarily associated in its naturalsource.

Methods of Treating Diseases Associated with Reduced MIF Expression

In certain embodiments, the invention features methods of increasingAMPK activity and glucose uptake in a subject in need thereof,comprising administering to a subject in need thereof a therapeuticallyeffective amount of one or more MIF agonists. In some embodiments, theinvention comprises administering to a subject having, or at risk ofdeveloping, tissue ischemia. In some embodiments, the subject suffersfrom a condition in which augmenting AMPK activity provides favorablemetabolic effects. In some embodiments the AMPK activation providesprotective effects to prevent cellular tissue injury. In anotherembodiment, the invention comprises administering to a subject having,or at risk of developing, type 2 diabetes.

Conditions that are associated with a need for increasing AMPK activityinclude without limitation, conditions that are associated withinadequate blood flow. Such conditions include, without limitation,myocardial ischemia and infarction, transient ischemic attack, stroke,hypoxia, tachycardia, atherosclerosis, hypotension, tissue ischemia fromthromboembolism, compression of blood vessels, foreign bodies in theblood circulation, sickle cell disease, cerebrovascular injury andperipheral artery occlusive disease.

The methods and compositions described herein also can be used prior totreatments or conditions that may result in inadequate blood flow. Whereischemia is anticipated in a subject with reduced MIF expression, it isparticularly advantageous to pretreat the subject with a MIF agonist.For example, the methods and compositions described herein may be usefulfor organ preservation or prior to or during organ transplantation.Prophylactic treatment with a MIF agonist also is advantageous insubjects suffering from conditions such as unstable angina, transientischemic attack and the like, in which reduced blood flow and/or tissueischemia is anticipated.

In another embodiment, the methods and compositions described herein canbe used in connection with type 2 diabetes or other diseases orconditions that may benefit from increased glucose uptake into thecells.

The methods and compositions described herein are useful for increasingAMPK activity in any cells, tissues, or organs of the body. For example,the methods and compositions described herein may be used to treatconditions that occur in cardial and skeletal muscle, the heart, brain,liver, kidney, and lungs. In particular, the methods and compositionsdescribed herein may be used to prevent cardiac injury and dysfunctionischemia and to prevent loss of viability or function of organs fortransplant.

In another embodiment, the methods and compositions described herein canbe used in connection with type 2 diabetes or other diseases orconditions that may benefit from increased glucose uptake into thecells, or where increased AMPK activation can delay or reduce adversemetabolic effects or tissue injury.

In various embodiments, MIF agonists can be, for example, drugs,metabolites, intermediates, cofactors, transition state analogs, ions,metals, toxins and natural and synthetic polymers (e.g., proteins,peptides, nucleic acids, polysaccharides, glycoproteins, hormones,receptors and cell surfaces such as cell walls and cell membranes.Agents may also comprise alcohols, alkyl halides, amines, amides,esters, aldehydes, ethers and other classes of organic agents. ExemplaryMIF agonists are known in the art. Further, certain MIF agonists aredescribed infra and supra.

In some embodiments the subjects carry a MIF gene polymorphism thatresults in reduced MIF expression and/or activity.

Prognostic and Diagnostic Methods

In one aspect, the invention provides methods for identifying subjectsin whom it would be particularly advantageous to administer one or moreMIF agonists to increase AMPK activity. The methods comprise genotypingthe subject for the presence of a polymorphism associated with reducedMIF expression.

Polymorphisms in the structure of the promoter region of the MIF geneare known to affect the level of MIF expression (Baugh et al. (2002)Genes Immun. 3:170-176 and De Benedetti et al. (2003) Arthritis & Rheum48:1398-1407).

A polymorphism associated with altered MIF expression may be any geneticalteration that modifies or correlates with the expression or activityof MIF. Polymorphisms associated with reduced MIF expression include,without limitation, the presence of five or fewer GATT repeats in the−794 region of one or both alleles of the MIF promoter, or a guanine atposition −173 of one or both alleles of the MIF promoter. Polymorphismsassociated with increased MIF expression include, without limitation,the presence of six, seven or eight CATT repeats in the −794 region ofboth alleles of the MIF promoter or the presence of a nucleotide otherthan guanine at position −173 of both alleles of the MIF promoter. In aspecific embodiment, a polymorphism associated with increased MIFexpression is the presence of cytosine (C) at position −173 of bothalleles of the MIF gene. In general, the greater the number of CATTrepeats present in the −794 region of the MIF promoter, the greater theexpression and/or activity of MIF. The above polymorphisms areillustrative of polymorphisms that may be associated with altered MIFexpression. Nevertheless, subjects in particular need of MIF agonisttherapy include those with any polymorphism that results in reduced MIFexpression or activity. As an illustrative embodiment, polymorphismsconsisting of a G/A or G/T nucleotide change at position −173 of the MIFpromoter may be associated with high or reduced MIF expression. Inanother illustrative embodiment, the presence of two, three, four, nine,ten, eleven or twelve or more CATT repeats in the −794 region of the MIFpromoter may be associated with increased or decreased MIF expression.Methods of genotyping a subject for the presence of a polymorphism(including single nucleotide polymorphisms and microsatellite repeats)in a gene are well known and routinely used in the art. Exemplarymethods of genotyping a subject for the presence of a polymorphism inthe MIF gene are described below.

In one embodiment, the methods of the invention are useful for selectinga subject for treatment with a MIF agonist, wherein the subject is inneed or may imminently be in need of increased AMPK activity. Forexample, the methods of the invention are useful for selecting a subjectfor treatment with a MIF agonist, wherein the subject has or is at riskof developing a condition associated with inadequate blood flow.Alternatively, the methods of the invention are useful for selecting asubject for treatment with a MIF agonist, wherein the subject has or isat risk of developing type 2 diabetes or any other condition that maybenefit from increased glucose uptake into cells. Such methods comprisegenotyping the subject to detect the presence of a polymorphismassociated with reduced MIF expression, wherein a subject having apolymorphism associated with reduced MIF expression is identified as acandidate for treatment with a MIF agonist.

In other embodiments, genotyping subjects to identify reduced MIFexpressers provides a means of predicting the potential severity ofischemic damage in a subject and for identifying subjects at particularrisk for ischemia/hypoxia-related injury.

Genotyping Assays

Certain aspects of the invention comprise genotyping a subject for thepresence of a polymorphism associated with reduced MIF expression. Anyassay that permits detection of a polymorphism in the MIF gene (which isused herein to include the MIF coding region and the MIF promoterregion) may be used in the claimed methods. The preferred method fordetecting a polymorphism will depend, in part, upon the molecular natureof the polymorphism. For example, certain methods may be amenable to thedetection of insertions, deletions, substitutions, repeats, or singlenucleotide polymorphisms (SNPs). Such assays are well known in the artand may encompass, for example, DNA sequencing, hybridization, ligation,or primer extension methods.

In one embodiment, genotyping a subject may comprise contacting a sampleobtained from the subject with a polynucleotide probe that hybridizesspecifically to a sequence comprising a polymorphism associated withaltered MIF expression, and, determining whether hybridization occurs.The polynucleotide probe can be engineered to hybridize specifically toa sequence comprising a polymorphism associated with reduced MIFexpression, but not to a sequence comprising a polymorphism associatedwith increased MIF expression. Hybridization of the probe to the DNA inthe sample indicates whether the subject comprises a polymorphismassociated with reduced MIF expression. In certain embodiments, methodsfor genotyping subject for the presence of a polymorphism that isassociated with, altered MIF expression further comprises contacting asample obtained from the subject with a control polynucleotide probe. Acontrol polynucleotide probe will not, for example, hybridizespecifically to a sequence comprising a polymorphism associated withreduced MIF expression. The polynucleotide probe may comprisenucleotides that are fluorescently, radioactively, or chemically labeledto facilitate detection of hybridization.

Hybridization may be performed and detected by standard methods known inthe art, such as by Northern blotting, Southern blotting, fluorescent insitu hybridization (FISH), or by hybridization to polynucleotidesimmobilized on a solid support, such as a DNA array or microarray. Arrayelements may comprise any polynucleotide, including genomic DNA, cDNA,synthetic DNA or other types of nucleic acid array elements.

In one embodiment, the probe is a DNA probe that is immobilized on asolid support, such as a DNA array or microarray. In one embodiment, theprobe is from about 8 nucleotides to about 500 nucleotides.

In another embodiment, a subject is genotyped for the presence of apolymorphism associated with altered MIF expression by hybridization toa DNA array or microarray, by incorporation of biotinylated primersfollowed by avidin-enzyme conjugate detection, or by incorporation of³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, intothe target polynucleotides (e.g., a polynucleotide that may include apolymorphism that is associated with altered MIF expression).Hybridization may be detected, for example, by measuring the intensityof the labeled probe remaining on a DNA array after washing.

Methods of detecting a polymorphism associated with reduced MIFexpression may include amplification of a region of DNA that comprises apolymorphism that is associated with reduced MIF expression. Any methodof amplification may be used. In one specific embodiment, a region ofDNA comprising the variation is amplified by using polymerase chainreaction (PCR). PCR was initially described by Mullis (See e.g., U.S.Pat. Nos. 4,683,195 4,683,202, and 4,965,188, herein incorporated byreference), which describes a method for increasing the concentration ofa region of DNA, in a mixture of genomic DNA, without cloning orpurification. Other PCR methods may also be used for nucleic acidamplification, including but not limited to RT-PCR, quantitative PCR,real time PCR, Rapid Amplified Polymorphic DNA Analysis, RapidAmplification of cDNA Ends (RACE), rolling circle amplification, ormultiple displacement amplification. For example, polynucleotide primersthat flank the MIF gene (including the MIF promoter) are combined with aDNA mixture. The mixture also includes the necessary amplificationreagents (e.g., deoxyribonucleotide triphosphates, buffer, etc.)necessary for the thermal cycling reaction. According to standard PCRmethods, the mixture undergoes a series of denaturation, primerannealing, and polymerase extension steps to amplify the region of DNAthat comprises a polymorphism that is associated with reduced MIFexpression. The length of the amplified region of DNA is determined bythe relative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. For example,hybridization of the primers may occur such that the ends of the primersproximal to the variation are separated by 1 to 10,000 base pairs (e.g.,10 base pairs (bp) 50 bp, 200 bp, 500 bp, 1,000 bp, 2,500 bp, 5,000 bp,or 10,000 bp).

In other embodiments, methods for genotyping a subject for the presenceof a polymorphism that is associated with reduced MIF expressioncomprise: (a) contacting a sample obtained from the subject with a pairof amplification primers, wherein said primers are capable of amplifyinga portion of the MIF promoter comprising a polymorphism associated withreduced MIF expression; (b) amplifying DNA in the sample, therebyproducing amplified DNA; and (c) determining whether the amplified DNAcomprises a polymorphism associated with reduced MIF expression, therebygenotyping the subject for the presence of a polymorphism associatedreduced MIF expression. The step of determining whether the amplifiedDNA comprises a polymorphism associated with reduced MIF expression canbe carried out using any method known in the art and/or describedherein. The method may further comprise sequencing the amplified DNA.

In one embodiment, the presence of a polymorphism associated withreduced MIF expression is detected and/or determined by DNA sequencing.Any of a variety of sequencing reactions known in the art can be used todirectly sequence the allele. Examples of sequencing reactions includethose based on techniques developed by Maxim and Gilbert ((1977) Proc.Natl. Acad. Sci. USA 74:560) or Sanger (Sanger et al (1977) Proc. Nat.Acad. Sci. USA 74:5463), DNA sequence determination may be performed bystandard methods such as dideoxy chain termination technology andgel-electrophoresis, or by other methods such as by pyrosequencing(Biotage AB, Uppsala, Sweden). For example, DNA sequencing by dideoxychain termination may be performed using unlabeled primers and labeled(e.g., fluorescent or radioactive) terminators. Alternatively,sequencing may be performed using labeled primers and unlabeledterminators. It is also contemplated that any of a variety of automatedsequencing procedures may be utilized when performing the subject assays(see, for example Biotechniques (1995) 19:448), including sequencing bymass spectrometry (see, for example PCT publication WO 94/16101; Cohenet al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993)Appl. Biochem. Biotechnol. 38:441-159). The nucleic acid sequence of theDNA in the sample can be studied to determine whether a polymorphismassociated with reduced MIF expression is present. It will be evident toone of skill in the art that, for certain embodiments, the occurrence ofonly one, two or three of the nucleic acid bases need be determined inthe sequencing reaction. For instance, A-track or the like, e.g., whereonly one nucleic acid is detected, can be carried out.

In another embodiment, the presence of a polymorphism associated withreduced MIF expression is detected and/or determined using FISH. Forexample, a probe that specifically hybridizes to a sequence comprising apolymorphism associated with reduced MIF expression is hybridized to asubject's genomic DNA by FISH. FISH can be used, for example, inmetaphase cells, to detect a deletion or repeat region in genomic DNA.Genomic DNA is denatured to separate the complimentary strands withinthe DNA double helix structure. The polynucleotide probe of theinvention is then added to the denatured genomic DNA. In a specificembodiment, a probe that specifically hybridizes to a sequencecomprising a polymorphism associated with reduced MIF expression isused. Accordingly, if a polymorphism associated with reduced MIFexpression is present, the probe will hybridize to the genomic DNA. Theprobe signal (e.g., fluorescence) can then be detected through afluorescent microscope for the presence of absence of signal. Thepresence of signal, therefore, indicates the presence of a polymorphismassociated with reduced MIF expression.

In another embodiment, the presence of a polymorphism associated withaltered MIF expression is detected and/or determined by primer extensionwith DNA polymerase. In one embodiment, a polynucleotide primer of theinvention hybridizes immediately adjacent to the polymorphism. A singlebase sequencing reaction using labeled dideoxynucleotide terminators maybe used to detect the polymorphism. In one embodiment, the presence of apolymorphism associated with reduced MIF expression will result in theincorporation of the labeled terminator, whereas the absence of apolymorphism associated with reduced MIF expression will not result inthe incorporation of the terminator. In another embodiment, thedideoxynucleotides may be labeled (e.g., fluorescently, radioactively,chemically, etc.) and the polymorphism is detected by detecting theincorporation of the labeled dideoxynucleotides during or after primerextension. In another embodiment, a polynucleotide primer of theinvention hybridizes specifically to a sequence comprising apolymorphism associated with reduced MIF expression. The presence of apolymorphism will result in primer extension, whereas the absence of apolymorphism will not result in primer extension. The primers and/ornucleotides may further include fluorescent, radioactive, or chemicalprobes. A primer labeled by primer extension may be detected bymeasuring the intensity of the extension product, such as by gelelectrophoresis, mass spectrometry, or any other method for detectingfluorescent, radioactive, or chemical labels.

In another embodiment, the presence of a polymorphism associated withaltered MIF expression is detected and/or determined by ligation. In oneembodiment, a polynucleotide primer hybridizes specifically to asequence comprising a polymorphism associated with reduced MIFexpression. A second polynucleotide that hybridizes to a region of theMIF gene immediately adjacent to the first primer is also provided. One,or both, of the polynucleotide primers may be fluorescently,radioactively, or chemically labeled. Ligation of the two polynucleotideprimers will occur in the presence of DNA ligase if a polymorphismassociated with reduced MIF expression is present. Ligation may bedetected by gel electrophoresis, mass spectrometry, or by measuring theintensity of fluorescent, radioactive, or chemical labels.

For example, identification of a polymorphism can be carried out usingan oligonucleotide ligation assay (OLA), as described, e.g., in U.S.Pat. No. 4,998,617 and in Landegren, U. et al. ((1988) Science241:1077-1080). The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990)Proc. Natl. Acad. Sci. USA 87:8923-27). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA.

Several techniques based on this OLA method have been developed and canbe used to identify a polymorphism. For example, U.S. Pat. No. 5,593,826discloses an OLA using an oligonucleotide having 3′-amino group and a5′-phosphorylated oligonucleotide to forth a conjugate having aphosphoramidate linkage. In another variation of OLA described in Tobeet al. ((1996) Nucleic Acids Res. 24: 3728), OLA combined with PCRpermits typing of two alleles in a single microtiter well. By markingeach of the allele-specific primers with a unique hapten, i.e.digoxigenin and fluorescein, each OLA reaction can be detected by usinghapten specific antibodies that are labeled with different enzymereporters, alkaline phosphatase or horseradish peroxidase. This systempermits the detection of polymorphisms using a high throughput formatthat leads to the production of two different colors.

In another embodiment, the presence of a polymorphism associated withaltered MIF expression is detected and/or determined by single-baseextension (SBE). For example, a fluorescently-labeled primer that iscoupled with fluorescence resonance energy transfer (FRET) between thelabel of the added base and the label of the primer may be used.Typically, the method, such as that described by Chen et al., (PNAS94.10756-61 (1997), incorporated herein by reference) uses alocus-specific polynucleotide primer labeled on the 5′ terminus with5-carboxyfluorescein (FAM). This labeled primer is designed so that the3′ end is immediately adjacent to the polymorphic site of interest. Thelabeled primer is hybridized to the locus, and single base extension ofthe labeled primer is performed with fluorescently labeleddideoxyribonucleotides (ddNTPs) in dye-terminator sequencing fashion,except that no deoxyribonucleotides are present. An increase influorescence of the added ddNTP in response to excitation at thewavelength of the labeled primer is used to infer the identity of theadded nucleotide.

In certain embodiments, a polymorphism that is associated with alteredMIF expression may be detected using single-strand conformationpolymorphism analysis, which identifies base differences by alterationin electrophoretic migration of single stranded PCR products, asdescribed in Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989).Amplified PCR products can be generated as described above, and heatedor otherwise denatured, to form single stranded amplification products.Single-stranded nucleic acids may refold or form secondary structureswhich are partially dependent on the base sequence. The differentelectrophoretic mobilities of single-stranded amplification products canbe related to base-sequence differences between alleles of targetsequences.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately adjacent 3′ tothe polymorphic site is permitted to hybridize to a target moleculeobtained from a subject. If the polymorphic site on the target moleculecontains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087).As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92115712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Recently, several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA™ in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

For SNPs that produce premature termination of protein translation, theprotein truncation test (PIT) offers an efficient diagnostic approach(Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21; van der Luijt, et.al., (1994) Genomics 20:1-4). For PTT, RNA is initially isolated fromavailable tissue and reverse-transcribed, and the segment of interest isamplified by PCR. The products of reverse transcription PCR are thenused as a template for nested PCR amplification with a primer thatcontains an RNA polymerase promoter and a sequence for initiatingeukaryotic translation. After amplification of the region of interest,the unique motifs incorporated into the primer permit sequential invitro transcription and translation of the PCR products. Upon sodiumdodecyl sulfate-polyacrylamide gel electrophoresis of translationproducts, the appearance of truncated polypeptides signals the presenceof a mutation that causes premature termination of translation. In avariation of this technique, DNA (as opposed to RNA) is used as a PCRtemplate when the target region of interest is derived from a singleoxen.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of subject tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, N.Y.).

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetraoxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labeled) RNA or DNA containing the one of thepolymorphic alleles with the sample. The double-stranded duplexes aretreated with an agent that cleaves single-stranded regions of the duplexsuch as which will exist due to base pair mismatches between the controland sample strands. For instance, RNA/DNA duplexes can be treated withRNase and DNA/DNA hybrids treated with S1 nuclease to enzymaticallydigest the mismatched regions. In other embodiments, either DNA/DNA orRNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxideand with piperidine in order to digest mismatched regions. Afterdigestion of the mismatched regions, the resulting material is thenseparated by size on denaturing polyacrylamide gels to determine thesite of mutation. See, for example, Cotton et al (1988) Proc. Natl AcadSci USA 85:4397; and Saleeba at al (1992) Methods Enzymol. 217:286295.In a preferred embodiment, the control DNA or RNA can be labeled fordetection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes). For example, the mutYenzyme of E. coli cleaves A at G/A mismatches and the thymidine DNAglycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.(1994) Carcinogenesis 15:1657-1662).

Commercial assays, such as the Taqman assay (Applied Biosystems, Fostercity, CA), may also be used for genotyping a subject for the presence ofa polymorphism that is associated with altered MIF expression.Genotyping and uses therefore are shown in U.S. Ser. Nos. 10/442,021,10/013,598 (U.S. Patent Application Publication 20030036069), and U.S.Pat. Nos. 5,925,525, 6,268,141, 5,856,092, 6,267,152, 6,300,063,6,525,185, 6,632,611, 5,858,659, 6,284,460, 6,361,947, 6,368,799,6,673,579 and 6,333,179.

Polynucleotides used in any of the methods of the invention, includingprobes and primers, can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. Polynucleotides of the invention can be modified at thebase moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Polynucleotideprobes of the invention may hybridize to a segment of target DNA suchthat the variation aligns with a central position of the probe, or thevariation may align with a terminal position of the probe.

Standard instrumentation known to those skilled in the art can be usedfor the amplification and detection of amplified DNA. For example, awide variety of instrumentation has been developed for carrying outnucleic acid amplifications, particularly PCR, e.g. U.S. Pat. No.5,038,852 (computer-controlled thermal cycler); Wittwer et al., NucleicAcids Research, 17: 4353-4357 (1989) (capillary tube PCR); U.S. Pat. No.5,187,084 (air-based temperature control); Garner et al, Biotechniques,14: 112-115 (1993)(high-throughput PCR in 864-well plates);International application No. PCT/US93/04039 (PCR in micro-machinedstructures); European patent application No. 90301061.9 (publ. No.0381501 A2) (disposable, single use PCR device), and the like. Incertain embodiments, the invention described herein utilizes real-timePCR or other methods known in the art such as the Taqman assay.

MIF Polymorphism Detection Using Immobilized Probes

In certain embodiments, a polymorphism in the MIF gene that isassociated with altered MIF expression may be detected usingpolynucleotide probes that have been immobilized on a solid support orsubstrate. Immobilized polynucleotide probes hybridize to a region ofthe MIF gene (including the promoter region of the MIF gene) thatcomprises a polymorphism that is associated with altered MIF expression.The present invention may employ any solid substrate known in the art,including arrays in some preferred embodiments. Methods and techniquesapplicable to polymer (including protein) array synthesis have beendescribed in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos.5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783,5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215,5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734,5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324,5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860,6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCTApplications Nos. PCT/US99/00730 (International Publication No. WO99/36760) and POT/US01/04285 (International Publication No. WO01/58593), which are all incorporated herein by reference in theirentirety for all purposes. Patents that describe synthesis techniques inspecific embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205,6,262,216, 6,310,189, 5,889,165, and 5,959,098.

In a specific embodiment, the invention provides a solid support orsubstrate for simultaneously genotyping a microsatellite repeat and aSNP in the MIF gene (including the promoter region). Examples of solidsupports and substrates include, without limitation, a nucleic acidprobe array (e.g., a chip, a microarray, or an array), a nitrocellulosefilter, a microwell, a bead, a sample tube, a microscope slide, amicrofluidics device, and the like. The solid support may be made ofvarious materials, including paper, cellulose, nylon, polystyrene,polycarbonate, plastics, glass, ceramic, stainless steel, or the like.The solid support may have a rigid or semi-rigid surface, and may bespherical (e.g., bead) or substantially planar (e.g., flat surface) withappropriate wells, raised regions, etched trenches, or the like. Thesolid support may also include a gel or matrix in which nucleic acidsmay be embedded or fibers or any solid support comprising bound nucleicacids. The solid support comprises at least two polynucleotide probesthat are complementary to one or more polymorphisms associated withaltered MIF expression. In a preferred embodiment, at least one of theprobes detects a microsatellite repeat associated with altered MIFexpression and at least one of the other probes detects a SNP associatedwith altered MIF expression. Hybridization to the polynucleotide probescan be detected using any detection method. In one embodiment,hybridization may be detected by the naked eye, without the aid ofinstruments for visualizing hybridization. Platforms for detection bythe naked eye include thin-film technologies such as those described inDenison et al., Expert Rev. Mol. Diagn. 6:89-99 (2006); Ostroff et al.,Clinical Chemistry 45:1659-1664 (1999) and Zhong et al., PNAS100:11559-11564 (2003), which are hereby incorporated by reference.

Thus, in a preferred embodiment, the invention provides the use of thinfilm technology to simultaneously genotype a microsatellite repeat and aSNP in the MIF gene. For example, in one embodiment, the inventionprovides the use of a thin film chip or microarray. Thin-film technologypermits the visual detection of nucleic acid targets with the unaidedeye. The assay is inexpensive, robust, highly specific, rapid and easyto use, thus permitting its implementation in rural settings withlimited technology. See Jenison et al. (2006) Expert Rev. Mol. 6:89-99.

Thin film technology is capable of generating a visual signal by thedirect interaction of light with thin films formed on a solid surface(e.g., a silicon surface). The surface is constructed to beantireflective to specific wavelengths of light by the addition ofantireflective coatings that create destructive interference. When lightreflected from the surface-thin-film interface is out of phase withlight reflected from the air-thin-film interface, specific, wavelengthsof light are eliminated from the reflected light by destructiveinterference, creating a characteristic surface color. Optical thicknessof the thin filth, which is a function of both refractive index andphysical thickness, determines which wavelengths of light areantireflected. Changes in the optical thickness of the thin film willresult in a visible color change on the surface, once it is dried. Thisoptical principle has been exploited to configure biologic assays onoptical surfaces that transduce a thickness change into a surface colorchange that is a direct measure of interactions between target moleculesin solution and capture molecules on the surface of the chip. The methodis sensitive to thickness changes in the angstrom range, translatinginto highly sensitive detection of target molecules in very rapid assayformats.

By amplifying molecular interactions, the increased mass deposited onthe surface of a thin film chip can be visually detected. Thin filmformation can be accomplished by a variety of signal amplificationtechniques, such as by the enzymatic turnover of precipitatingsubstrates. For the detection of nucleic acid sequences, thin filmdevelopment may utilize, for example, the detection of biotin-labeledprobes by binding of an antibiotin antibody conjugated to horseradishperixidase (HRP). In the presence of a precipitating substrate for HRP,an enhanced molecular thin film is deposited onto the surface of thesolid substrate. Control of the reflective properties to create, forexample, a gold-colored surface is achieved by the coating of surfaceswith optical layers of defined refractive index and thickness, usingwell-established semiconductor processes. Details of the preparation ofthe optically coated surfaces have been described previously. See, forexample, Jenison et al. (2001). Biotech. 19:62-65. Briefly, the basesurface of the chip is crystalline silicon, which provides a highlyreflective, inert and molecularly flat surface to which theantireflective coating (silicon nitride) is applied by vapor deposition(e.g., to a thickness of 475 angstroms). An attachment layer, such asT-structure aminoalkyl polydimethy siloxane (TSPS) can be coated on thesurface to provide better immobilization of biological materials, suchas nucleic acid capture probes or antibodies.

In one embodiment, a solid support as described above (such as a chip ormicroarray) comprises at least one probe that hybridizes specifically toa sequence comprising a guanine or to a sequence comprising a cytosineat position −173 of the MIF promoter.

In another embodiment, a solid support as described above (such as achip or micro array) comprises at least one probe that hybridizesspecifically to a sequence comprising a sequence selected from the groupconsisting of SEQ ID NO: 1 (CATTCATTCATTCATTCATT), SEQ ID NO: 2(CATTCATTCATTCATTCATTCATT), SEQ ID NO: 3 (CATTCATTCATTCATTCATTCATTCATT),and SEQ ID NO: 4 (CATTCATTCATTCATTCATT CATTCATTCATT).

In another embodiment, a solid support as described above (such as achip or micro array) comprises: (a) at least one probe that hybridizesspecifically to a sequence comprising a guanine or a cytosine atposition −173 of the MIF promoter; and, (b) at least one probe thathybridizes specifically to a sequence comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,and SEQ ID NO: 4.

In another embodiment, a solid support as described above (such as achip or microarray) comprises a probe that hybridizes specifically to asequence comprising a guanine at position −173 of the MIF promoter andanother probe that hybridizes specifically to a sequence comprising acytosine at position −173 of the MIF promoter.

In another embodiment, a solid support as described above (such as achip or microarray) comprises: (a) a probe that hybridizes specificallyto a sequence comprising SEQ ID NO: 1; (b) a probe that hybridizesspecifically to a sequence comprising SEQ ID NO: 2; (c) a probe thathybridizes specifically to a sequence comprising SEQ ID NO: 3; and, (d)a probe that hybridizes specifically to a sequence comprising SEQ ID NO:4.

In another embodiment, a solid support as described above (Rich as achip or microarray) comprises: (a) a probe that hybridizes specificallyto a sequence comprising a guanine at position −173 of the MIF promoter;(b) a probe that hybridizes specifically to a sequence comprising acytosine at position −173 of the MIF promoter; (c) a probe thathybridizes specifically to a sequence comprising SEQ ID NO: 1; (d) aprobe that hybridizes specifically to a sequence comprising SEQ ID NO:2; (e) a probe that hybridizes specifically to a sequence comprising SEQID NO: 3; and, (f) a probe that hybridizes specifically to a sequencecomprising SEQ ID NO: 4.

In one embodiment, the invention provides a method of determining theMIF genotype of a subject comprising contacting a solid substrate asdisclosed herein with a sample obtained from the subject and determiningthe MIF genotype of the subject. The sample may be amplified prior tocontacting the sample with the solid substrate disclosed herein. In oneembodiment, the invention provides a method of determining the MIFgenotype of a subject comprising: (a) amplifying a portion of the MIFgene comprising a polymorphism associated with altered MIF expression;(b) contacting a solid substrate as disclosed herein with the amplifiedportion; and, (c) determining whether the subject comprises apolymorphism associated with increased MIF expression or whether thesubject comprises a polymorphism associated with reduced MIF expression,thereby determining the MIF genotype of the subject.

Other Genotyping Methods

Moreover, the genotyping methods disclosed herein can be substituted bythe use of other methods that can establish whether a subject expressesMIF at reduced or increased levels. Such methods are therefore usefulfor identifying a subject particularly in need of a MIF agonist orsusceptible to injury associated with reduced blood flow.

For example, the MIF protein levels can be measured in a subject andcompared to the MIF protein levels in a subject with a genotype that isassociated with reduced MIF expression (e.g., a guanine at position −173of one or both alleles of the MIF promoter or five CATT repeats in the−794 region of one or both alleles of the MIF promoter). Alternatively,MIF protein levels can be measured in a subject and compared to the MIFprotein levels in a subject with a genotype that is associated withincreased MIF expression (e.g., a cytosine at position −173 of bothalleles of the MIF promoter and/or six or more CATT repeats in the −794region of both alleles of the MIF promoter).

Standard methods for measuring protein levels are known in the art. See,for example, Current Protocols in Molecular Biology, eds. Ausubel etal., John Wiley & Sons: 1992. For example, MIF protein levels can bemeasured by measuring the amount of light aborbance in a sample of theprotein. Alternatively, MIF protein levels can be measured using anagent that binds to MIF protein, such as an antibody, an aptamer, asmall molecule, another protein or an enzyme. Binding of the agent toMIF can be detected by the use of a signal (e.g., fluorescent,colorimetric, radioactive, chemical, or enzymatic), or may be detectedby a chemical or enzymatic reaction. Other methods of measuring proteinlevels may include mass spectrometry, surface plasmon resonance or usingprotein chips.

The Use of MIF Agonists for Treatment

In one embodiment, the invention provides a method of increasing AMPKactivity comprising administering to a subject a therapeuticallyeffective amount of a MIF agonist. In some embodiments, the subject hasa genotype that is associated with reduced MIF expression. In someembodiments, the subject has or is at risk of having a condition inwhich increased glucose uptake is desirable. Such conditions includehypoxia, especially hypoxia resulting from tissue ischemia. In variousembodiments, the tissue ischemia is associated with acute coronarysyndromes such as myocardial infarction, coronary revascularization(such as coronary bypass surgery and coronary angioplasty/stentplacement), stroke, renal, retinal, mesenteric or limb ischemia due tovascular occlusion, organ transplant surgery (for maintaining viabilityand function of the transplanted organ), ischemia associated withvascular surgery, including hypothermic arrest and vascularcross-clamping.

In another embodiment, the condition is type 2 diabetes.

In one embodiment, a MIF agonist is administered to a subject prior tothe observation of any symptoms indicating that increased AMPK activityor glucose uptake is desirable, such as signs of cell or tissue injury,loss of function or cell death. For example, a MIF agonist may beadministered to an organ donor prior to organ removal. Alternatively, aMIF agonist may be administered as a prophylactic measure to prevent,e.g., ischemic injury or to patients at risk of experiencing decreasedblood flow (e.g., patients at risk for ischemia). A MIF agonist also maybe used to treat an organ prior to transplantation in a recipient.

The methods described herein may also comprise the administration of aMIF agonist with one or more additional therapeutic agents. In variousembodiments, the therapeutic agents or agents that increase AMPKactivity, stimulate blood flow, or increase glucose uptake. Such agentsinclude but are not limited to metformin, rosiglitazone, pioglitazone,leptin, adiponectin and acadesine.

In other embodiments, a MIF agonist may be co-administered eitherseparately or in the same dosage form with a fatty acid oxidationinhibitor. Such inhibitors are known in the art and include, e.g.,ranolazine.

MIF Angonists

As described above any agent that mimics, activates, stimulates,potentiates or increases the biological activity of MIF can be used as aMIF agonist.

MIF agonists include isolated or recombinant nucleic acids that encodeMIF proteins or fragments thereof; isolated or recombinant MIFpolypeptides or fragments thereof; or other agents that mimic, activate,stimulate, potentiate or increase the biological activity of MIF.Examples of MIF agonists include, without limitation, agents thatincrease MIF mRNA or protein expression; agents that enhance CD44 mRNAor protein expression; agents that enhance CD74 mRNA or proteinexpression; agents that increase interaction between MIF, CD74 and CD44(e.g., bivalent antibodies that bind two out of three of MIF, CD74 andCD44, fusion proteins with CDR combinations that bind two out of threeof MIF, CD74 and CD44, and other agents that are identified by any ofthe screening methods described herein); agents that increaseinteraction between CD44 and CD74 (e.g., bivalent antibodies that bindCD74 and CD44, and other agents that are identified by any of thescreening methods described herein); and agents that increaseinteraction between MIF and CD74 (e.g., bivalent antibodies that bindMIF and CD74, and other agents that are identified by any of thescreening methods described herein). For example, agonist anti-CD74 oragonist anti-CD44 antibodies are useful as MIF pathway agonists. Otheragents that may be used, comprising the use of agents that modulate theinteraction between MIF, CD74 and CD44. Other agents that may be used tomimic, activate, stimulate, potentiate or increase the biologicalactivity of MIF include, without limitation, drugs, metabolites,intermediates, cofactors, transition state analogs, ions, metals,toxins, natural and synthetic polymers (e.g., proteins, peptides,nucleic acids, polysaccharides, glycoproteins, hormones, receptors andcell surfaces such as cell walls and cell membranes, and antisensenucleic acids. Agents may also comprise alcohols, alkyl halides, amines,amides, esters, aldehydes, ethers and other classes of organic agents.

In a preferred embodiment, the MIF agonist is recombinant MIF.

In one embodiment, the D-dopachrome tautomerase (DDT) protein, orfragments thereof, is used to agonize the biological activity of MIF. Inanother embodiment, an agonist of DDT is used to agonize the activity ofMIF. An exemplary nucleotide sequence that encodes DDT is found inGenBank Accession No. AH006997.

Aptamers and Small Molecules

Aptamers can also be used as MIF agonists. The present inventionprovides therapeutic aptamers that specifically bind to a MD polypeptideor a polypeptide that affects MIF expression or MIF biological function,thereby agonizing activity of MIF.

In one embodiment, a MIF agonist may be an aptamer that binds to a MIFpolypeptide and activates, stimulates or potentiates the activity ofsaid MIF polypeptide. In one embodiment, a MIF agonist may be an aptamerthat binds to a CD44 polypeptide and activates, stimulates orpotentiates the activity of said CD44 polypeptide. In one embodiment, aMD agonist may be an aptamer that binds to a CD74 polypeptide andactivates, stimulates or potentiates the activity of said CD74polypeptide.

An “aptamer” may be a nucleic acid molecule, such as RNA or DNA that iscapable of binding to a specific molecule with high affinity andspecificity (Ellington et al., Nature 346, 818-22 (1990); and Tuerk etal., Science 249, 505-10 (1990)). An aptamer will most typically havebeen obtained by in vitro selection for binding of a target molecule.For example, an aptamer that specifically binds to polypeptide importantfor the biological function of MIF (e.g., MD, CD74 or CD44) can beobtained by in vitro selection from a pool of polynucleotides forbinding to a polypeptide important for the biological function of MIF(e.g., MIF, CD74 or CD44). However, in vivo selection of an aptamer isalso possible. Aptamers have specific binding regions which are capableof forming complexes with an intended target molecule in an environmentwherein other substances in the same environment are not complexed tothe nucleic acid. The specificity of the binding is defined in terms ofthe comparative dissociation constants (Kd) of the aptamer for itsligand (e.g., a MIF polypeptide, or a polypeptide important for thebiological function of MD such as CD74 or CD44) as compared to thedissociation constant of the aptamer for other materials in theenvironment or unrelated molecules in general. A ligand (e.g., a MIF,CD74 or CD44 polypeptide) is one which binds to the aptamer with greateraffinity than to unrelated material. Typically, the Kd for the aptamerwith respect to its ligand will be at least about 10-fold less than theKd for the aptamer with unrelated material or accompanying material inthe environment. Even more preferably, the Kd will be at least about50-fold less, more preferably at least about 100-fold less, and mostpreferably at least about 200-fold less. An aptamer will typically bebetween about 10 and about 300 nucleotides in length. More commonly, anaptamer wilt be between about 30 and about 100 nucleotides in length.

Methods for selecting aptamers specific for a target of interest areknown in the art. For example, organic molecules, nucleotides, aminoacids, polypeptides, target features on cell surfaces, ions, metals,salts, saccharides, have all been shown to be suitable for isolatingaptamers that can specifically bind to the respective ligand. Forinstance, organic dyes such as Hoechst 33258 have been successfully usedas target ligands for in vitro aptamer selections (Werstuck and Green,Science 282:296-298 (1998)). Other small organic molecules likedopamine, theophylline, sulforhodamine B, and cellobiose have also beenused as ligands in the isolation of aptamers. Aptamers have also beenisolated for antibiotics such as kanamycin A, lividomycin, tobramycin,neomycin B, viomycin, chloramphenicol and streptomycin. For a review ofaptamers that recognize small molecules, see Famulok, Science 9:324-9(1999).

An aptamer of the invention can be comprised entirely of RNA. In otherembodiments of the invention, however, the aptamer can instead becomprised entirely of DNA, or partially of DNA, or partially of othernucleotide analogs. To specifically inhibit translation in vivo, RNAaptamers are preferred. Such RNA aptamers are preferably introduced intoa cell as DNA that is transcribed into the RNA aptamer. Alternatively,an RNA aptamer itself can be introduced into a cell.

Aptamers are typically developed to bind particular ligands by employingknown in vivo or in vitro (most typically, in vitro) selectiontechniques known as SELEX (Ellington et al., Nature 346, 818-22 (1990);and Tuerk et al., Science 249, 505.10 (1990)). Methods of makingaptamers are also described in for example, U.S. Pat. No. 5,582,981, PCTPublication No. WO 00/20040, U.S. Pat. No. 5,270,163, Lorsch andSzostak, Biochemistry, 33:973 (1994), Mannironi et al., Biochemistry36:9726 (1997), Blind, Proc. Nat'l. Acad. Sci. USA 96:3606-3610 (1999),Huizenga and Szostak, Biochemistry, 34; 656-665 (1995), PCT PublicationNos. WO 99/54506, WO 99/27133, WO 97/42317 and U.S. Pat. No. 5,756,291.

Generally, in their most basic form, in vitro selection techniques foridentifying aptamers involve first preparing a large pool of DNAmolecules of the desired length that contain at least some region thatis randomized or mutagenized. For instance, a common oligonucleotidepool for aptamer selection might contain a region of 20-100 randomizednucleotides flanked on both ends by an about 15-25 nucleotide longregion of defined sequence useful for the binding of PCR primers. Theoligonucleotide pool is amplified using standard PCR techniques,although any means that will allow faithful, efficient amplification ofselected nucleic acid sequences can be employed. The DNA pool is then invitro transcribed to produce RNA transcripts. The RNA transcripts maythen be subjected to affinity chromatography, although any protocolwhich will allow selection of nucleic acids based on their ability tobind specifically to another molecule (e.g., a protein or any targetmolecule) may be used. In the case of affinity chromatography, thetranscripts are most typically passed through a column or contacted withmagnetic beads or the like on which the target ligand has beenimmobilized. RNA molecules in the pool which bind to the ligand areretained on the column or bead, while nonbinding sequences are washedaway. The RNA molecules which bind the ligand are then reversetranscribed and amplified again by PCR (usually after elution). Theselected pool sequences are then put through another round of the sametype of selection. Typically, the pool sequences are put through a totalof about three to ten iterative rounds of the selection procedure. ThecDNA is then amplified, cloned, and sequenced using standard proceduresto identify the sequence of the RNA molecules which are capable ofacting as aptamers for the target ligand. Once an aptamer sequence hasbeen successfully identified, the aptamer may be further optimized byperforming additional rounds of selection starting from a pool ofoligonucleotides comprising the mutagenized aptamer sequence. For use inthe present invention, the aptamer is preferably selected for ligandbinding in the presence of salt concentrations and temperatures whichmimic normal physiological conditions.

The unique nature of the in vitro selection process allows for theisolation of a suitable aptamer that binds a desired ligand despite acomplete dearth of prior knowledge as to what type of structure mightbind the desired ligand.

The association constant for the aptamer and associated ligand ispreferably such that the ligand functions to bind to the aptamer andhave the desired effect at the concentration of ligand obtained uponadministration of the ligand. For in vivo use, for example, theassociation constant should be such that binding occurs well below theconcentration of ligand that can be achieved in the serum or othertissue. Preferably, the required ligand concentration for in vivo use isalso below that which could have undesired effects on the organism.

The present invention also provides small molecules and antibodies thatspecifically bind to a polypeptide important for the biological functionof MIF (e.g., MIF, CD74 or CD44), thereby agonizing the biologicalfunction of MIF. Examples of small molecules include, withoutlimitation, drugs, metabolites, intermediates, cofactors, transitionstate analogs, ions, metals, toxins and natural and synthetic polymers(e.g., proteins, peptides, nucleic acids, polysaccharides,glycoproteins, hormones, receptors and cell surfaces such as cell wallsand cell membranes).

Antibodies, Antibody Fragments and Other Fusion Proteins

Antibodies or fragments thereof specifically reactive with a polypeptidethat affects the expression or biological function of MIF may be used asMIF agonists. Antibodies or fragments thereof directed, for example, toMIF, CD44, CD74, and/or combinations thereof; may be agonists of theexpression or biological function of MM.

In certain embodiments, an antibody or fragment thereof that isspecifically reactive with a MIF polypeptide may be used as a MIFagonist to increase or activate the activity or a MIF polypeptide. Inone embodiment, a MIF agonist is an antibody, such as a bivalentantibody or a fragment thereof, that is able to bind MIF. In anotherembodiment, a MIF agonist is an antibody, such as a bivalent antibody ora fragment thereof; that is able to bind MIF and CD44. In anotherembodiment, a MIF agonist is an antibody, such as a bivalent antibody ora fragment thereof, that is able to CD44 and CD74. In anotherembodiment, a MILT agonist is an antibody, such as a bivalent antibodyor a fragment thereof, that is able to bind MIF and CD74

Methods of making antibodies are well known in the art. For example, byusing immunogens derived from a MIF polypeptide or a polypeptide thataffects the expression or biological function of MIF,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols (see, for example, Antibodies: A Laboratory Manualed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, suchas a mouse, a hamster or rabbit can be immunized with an immunogenicform of the MIF polypeptide, an antigenic fragment which is capable ofeliciting an antibody response, or a fusion protein. Such mammals canalso be immunized with an immunogenic form of a polypeptide that affectsthe expression or biological function of MIF. Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques well known in the art. An immunogenic portion of aMIF polypeptide or a polypeptide that affects the expression orbiological function of MIF can be administered in the presence ofadjuvant. The progress of immunization can be monitored by detection ofantibody titers in plasma or serum. Standard ELISA or other immunoassayscan be used with the immunogen as antigen to assess the levels ofantibodies.

Following immunization of an animal with an antigenic preparation of aMIF polypeptide or a polypeptide that affects the expression orbiological function of MIF, antisera can be obtained and, if desired,polyclonal antibodies can be isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fined by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a MIF polypeptide ora polypeptide that affects the expression or biological function of MIF.Monoclonal antibodies can be isolated from a culture comprising suchhybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a MIF polypeptide or apolypeptide that affects the expression or biological function of MIF.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab)₂ fragment can betreated to reduce disulfide bridges to produce Fab fragments.Antigen-binding portions may also be produced by recombinant DNAtechniques or by enzymatic or chemical cleavage of intact antibodies.Antigen-binding portions include, inter alia, Fab, Fab′, F(ab)₂, Fv,dAb, and complementarity determining region (CDR) fragments,single-chain antibodies (scFv), single domain antibodies, bispecificantibodies, chimeric antibodies, humanized antibodies, diabodies andpolypeptides that contain at least a portion of an immunoglobulin thatis sufficient to confer specific antigen binding to the polypeptide. Incertain embodiments, the antibody further comprises a label attachedthereto and able to be detected (e.g., the label can be a radioisotope,fluorescent compound, enzyme or enzyme co-factor).

In certain embodiments, an antibody of the invention is a monoclonalantibody, and in certain embodiments, the invention makes availablemethods for generating novel antibodies that bind specifically to MIFpolypeptides or to polypeptides that affect the expression or biologicalfunction of MIF. For example, a method for generating a monoclonalantibody that binds specifically to a MIF polypeptide, or to apolypeptide that affects the expression or biological function of MIF,may comprise administering to a mouse an amount of an immunogeniccomposition comprising the MIF polypeptide or the polypeptide thataffects the expression or biological function of MIF, effective tostimulate a detectable immune response, obtaining antibody-producingcells (e.g., cells from the spleen) from the mouse and fusing theantibody-producing cells with myeloma cells to obtain antibody-producinghybridomas, and testing the antibody-producing hybridomas to identify ahybridoma that produces a monocolonal antibody that binds specificallyto the MIF polypeptide or the polypeptide that affects the expression orbiological function of MIF. Once obtained, a hybridoma can be propagatedin a cell culture, optionally in culture conditions where thehybridoma-derived cells produce the monoclonal antibody that bindsspecifically to the MIF polypeptide or the polypeptide that affects theexpression or biological function of MIF. The monoclonal antibody may bepurified from the cell culture.

The tem “specifically reactive with” as used in reference to an antibodyis intended to mean, as is generally understood in the art, that theantibody is sufficiently selective between the antigen of interest(e.g., a MIF polypeptide or a polypeptide that affects the expression orbiological function of MIF) and other antigens that are not of interestthat the antibody is useful for, at minimum, detecting the presence ofthe antigen of interest in a particular type of biological sample. Incertain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

General Information Relating to Methods of Treatment Using MIF Agonists

The methods described herein for increasing AMPK activity may be usedprophylactically. Thus, in one embodiment, a composition comprising aMIF agonist is administered in an amount and dose that is sufficient todelay, slow, or prevent tissue injury associated with hypoxia includingtissue ischemia.

MIF agonists may be formulated with a pharmaceutically acceptablecarrier. For example, a MIF agonist can be administered alone or as acomponent of a pharmaceutical formulation (therapeutic composition). TheMIF agonist may be formulated for administration in any convenient wayfor use in human medicine.

In certain embodiments, the therapeutic methods of the invention includeadministering the composition topically, systemically, or locally. Forexample, therapeutic compositions of the invention may be formulated foradministration by, for example, injection (e.g., intravenously,subcutaneously, or intramuscularly), inhalation or insufflation (eitherthrough the mouth or the nose) or oral, buccal, sublingual, transdermal,nasal, or parenteral administration. The compositions described hereinmay be formulated as part of an implant or device. When administered,the therapeutic composition for use in this invention is in apyrogen-free, physiologically acceptable form. Further, the compositionmay be encapsulated or injected in a viscous form for delivery to thesite where the target cells are present. Techniques and formulationsgenerally may be found in Remington's Pharmaceutical Sciences, MeadePublishing Co., Easton, Pa. In addition to MIF agonists, therapeuticallyuseful agents may optionally be included in any of the compositionsdescribed herein. Furthermore, therapeutically useful agents may,alternatively or additionally, be administered simultaneously orsequentially with a MIF agonist according to the methods of theinvention.

In certain embodiments, compositions comprising a MIF agonist can beadministered orally, e.g., in the form of capsules, cachets, pills,tablets, lozenges (using a flavored basis, usually sucrose and acacia ortragacanth), powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouth washes and the like, each containing a predetermined amount ofan agent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more compositionscomprising a MIF agonist may be mixed with one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such asglycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrup; andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as water orother solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and atone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required. Theointments, pastes, creams and gels may contain, in addition to a MIFagonist, excipients, such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, snick acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to a MIF agonist, excipientssuch as lactose, talc, silicic acid, aluminum hydroxide, calciumsilicates, and polyamide powder, or mixtures of these substances. Sprayscan additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

In certain embodiments, pharmaceutical compositions suitable forparenteral administration may comprise a MIF agonist in combination withone or more pharmaceutically acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

A composition comprising a MIF agonist may also contain adjuvants, suchas preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of a MIF agonist. Such therapy would achieveits therapeutic effect by introduction of a polynucleotide sequence thatencodes a MIF agonist into cells or tissues to increase MIF expressionand/or activity. Delivery of MIF agonist polynucleotide sequences can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system. Targeted liposomes may also be usedfor the therapeutic delivery of MIF polynucleotide sequences.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus suchas a retrovirus. A retroviral vector may be a derivative of a murine oravian retrovirus. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Anumber of additional retroviral vectors can incorporate multiple genes.All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated.Retroviral vectors can be made target-specific by attaching, forexample, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing the polynucleotidethat encodes the MIF agonist.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for polynucleotides that encode MIFagonists is a colloidal dispersion system. Colloidal dispersion systemsinclude macromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see, e.g., Fraley, etal Trends Biochem. Sci. 6:77, 1981). Methods for efficient gene transferusing a liposome vehicle, are known in the art, see e.g., Mannino, atal., Biotechniques, 6:682, 1988. The composition of the liposome isusually a combination of phospholipids, usually in combination withsteroids, especially cholesterol. Other phospholipids or other lipidsmay also be used. The physical characteristics of liposomes depend onpH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

Moreover, a composition comprising a MIF agonist can consist essentiallyof the gene delivery system in an acceptable diluent, or can comprise aslow release matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery system can be producedintact from recombinant cells, e.g. retroviral packages, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system. In the case of the latter, methods ofintroducing the viral packaging cells may be provided by, for example,rechargeable or biodegradable devices. Various slow release polymericdevices have been developed and tested in vivo in recent years for thecontrolled delivery of drugs, including proteinaceousbiopharmaceuticals, and can be adapted for release of viral particlesthrough the manipulation of the polymer composition and form. A varietyof biocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of the viral particles by cellsimplanted at a particular target site. Such embodiments of the presentinvention can be used for the delivery of an exogenously purified virus,which has been incorporated in the polymeric device, or for the deliveryof viral particles produced by a cell encapsulated in the polymericdevice.

A person of ordinary skill in the art, such as a physician, is able todetermine the required amount to treat the subject. It is understoodthat the dosage regimen will be determined for an individual, takinginto consideration, for example, various factors that modify the actionof a MIF agonist, the severity of the condition associated with hypoxiaor tissue ischemia, route of administration, and characteristics uniqueto the individual, such as age, weight, and size. In one embodiment, thedosage can range from about 1.0 ng/kg to about 100 mg/kg body weight ofthe subject.

In certain embodiments, a composition comprising a MIF agonist fortopical, systemic or local administration can be administered in a rangefrom about 0.001% to about 3.0% (weight per volume or weight perweight), or from about 0.001% to about 0.01%, from about 0.01% to about0.025%, from about 0.025% to about 0.05%, from about 0.05% to about0.1%, from about 0.1% to about 0.25%, from about 0.25% to about 1.0%,from about 1.0% to about 2.0%, or from about 2.0% to greater than 3.0%,i.e., about 10% to about 10.0% or greater. In a specific embodiment, acomposition comprising a MIF agonist is administered in orange fromabout 0.25% to about 3.0%.

In certain embodiments, a composition comprising a MIF agonist isadministered in a range of from about 1 ng/ml to about 1 g/ml, or fromabout 1 ng/ml to about 10 ng/ml, from about 10 ng/ml to about 100 ng/ml,from about 100 ng/ml to about 1 mg/ml, from about 1 mg/ml to about 10mg/ml, from about 10 mg/ml to about 100 mg/ml or from about 100 mg/ml toabout 1 g/ml. In certain embodiments, a composition comprising a MIFagonist is administered in a range of from about 40 ng/ml to about 100ng/ml.

The volume of composition administered according to the methodsdescribed herein is also dependent on factors such as the mode ofadministration, quantity of the MIF agonist, age and weight of thepatient, and type and severity of the disease being treated. Forexample, if administered orally as a liquid, the liquid volumecomprising a composition comprising a MIF agonist may be from about 0.5milliliters to about 2.0 milliliters, from about 2.0 milliliters toabout 5.0 milliliters, from about 5.0 milliliters to about 10.0milliliters, or from about 10.0 milliliters to about 50.0 milliliters.If administered by injection, the liquid volume comprising a compositioncomprising a MIF agonist may be from about 5.0 microliters to about 50microliters, from about 50 microliters to about 250 microliters, fromabout 250 microliters to about 1 milliliter, from about 1 milliliter toabout 5 milliliters, from about 5 milliliters to about 25 milliliters,from about 25 milliliters to about 100 milliliters, or from about 100milliliters to about 1 liter.

The dose can be delivered continuously, or at periodic intervals (e.g.,on one or more separate occasions). Desired time intervals of multipledoses of a particular composition can be determined without undueexperimentation by one skilled in the art. For example, the compound maybe delivered hourly, daily, weekly, monthly, yearly (e.g. in a timerelease form) or as a one time delivery. If administered orally ortopically, such a preparation can be administered 1 to 6 times per dayfor a period of 1-4 weeks, 1-3 months, 3-6 months, 6-12 months, 1-2years, or more, up to the lifetime of the patient. If administered byinjection, MIF against compositions can be delivered one or more timesperiodically throughout the life of a patient. For example, a MIFagonist composition can be delivered once per year, once every 6-12months, once every 3-6 months, once every 1-3 months, once every 1-4weeks, one or more times per day. Alternatively, more frequentadministration may be desirable for certain conditions or disorders. Ifadministered by an implant or device, MIF agonist compositions can beadministered one time, or one or more times periodically throughout thelifetime of the patient as necessary.

Samples used in the methods described herein may comprise cells from theeye, ear, nose, throat, teeth, tongue, epidermis, epithelium, blood,tears, saliva, mucus, urinary tract, urine, muscle, cartilage, skin, orany other tissue or bodily fluid from which sufficient DNA, RNA orprotein can be obtained. In certain embodiments, samples used in themethods described herein comprise cells from a tracheal aspirate ornasal washing.

The sample should be sufficiently processed to render the DNA, RNA orprotein that is present in the sample available for assaying in themethods described herein. For example, samples may be processed suchthat DNA from the sample is available for amplification or forhybridization to another polynucleotide. The processed samples may becrude lysates where available DNA, RNA or protein is not purified fromother cellular material. Alternatively, samples may be processed toisolate the available DNA, RNA or protein from one or more contaminantsthat are present in its natural source. Samples may be processed by anymeans known in the art that renders DNA, RNA or protein available forassaying in the methods described herein. Methods for processing samplesmay include, without limitation, mechanical, chemical, or molecularmeans of lysing and/or purifying cells and cell lysates. Processingmethods may include, for example, ion-exchange chromatography, sizeexclusion chromatography, affinity chromatography, hydrophobicinteraction chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and imniunoaffinity purification withantibodies specific for particular epitopes of the polypeptide

Screening Assays

In certain aspects, the present invention relates to the use of MIF,CD44 and/or CD74 to identify agents that are agonists of AMPK activity.Agents identified through this screening can be tested in cells andtissues to assess their ability to modulate the biological activity ofAMPK in vivo or in vitro. In certain aspects, these agents can furtherbe tested in animal models to assess their ability to modulate thebiological activity of AMPK in vivo. The compounds identified in thesemethods can be used to increase AMPK activity as described herein. Themethods described herein are based on the discovery that CD44functionally interacts with MIF (Meyer-Siegler et al., BMC Cancer, 4:34(2004) and Meyer-Siegler et al., J Urol., 173:615-620 (2005)).

In one embodiment, the invention provides a method of identifyingpotential agonists of the biological activity of AMPK, comprising: (a)contacting a CD44 polypeptide, or a portion thereof, with a CD74polypeptide, or portion thereof, in the presence and absence of acandidate agent; and (b) comparing the interaction of the CD44 and CD74polypeptides in the presence of said candidate agent with theinteraction in the absence of said candidate agent. A candidate agentthat enhances the interaction of the CD44 polypeptide and the CD74polypeptide is thus identified as a potential agonist of AMPK biologicalfunction.

In another embodiment, the invention provides a method of identifyingpotential agonists of the biological activity of AMPK, comprising: (a)contacting a CD44 polypeptide or a portion thereof, with a MIFpolypeptide, or a portion thereof, and a CD74 polypeptide or a portionthereof, in the presence and absence of a candidate agent; and, (b)comparing the interaction of the CD44 polypeptide or portion thereof andthe MIF and CD74 polypeptides or portions thereof in the presence ofsaid candidate agent with the interaction in the absence of saidcandidate agent. A candidate agent that enhances the interaction of theCD44 polypeptide and the MIF and CD74 polypeptides is thus identified asa potential agonist of AMPK biological function.

The interaction between the agent and the subject polypeptide (e.g.,CD44, CD74, MIF, and/or MIF/CD74) may be covalent or non-covalent. Forexample, such interaction can be identified at the protein level usingin vitro biochemical methods, including photo-crosslinking, radiolabeledligand binding, and affinity chromatography (Jakoby W B et al., 1974,Methods in Enzymology 46:1). In certain cases, the agents may bescreened in a mechanism based assay, such as an assay to detect agentswhich bind to the subject polypeptide (e.g., CD44, CD74, and/orMIF/CD74). This may include a solid phase or fluid phase binding event.Alternatively, the gene or genes encoding one or more of the subjectpolypeptides can be transfected with a reporter system (e.g.,β-galactosidase, luciferase, or green fluorescent protein) into a celland screened against the library preferably by a high throughputscreening or with individual members of the library. Other mechanismbased binding assays may be used, for example, binding assays whichdetect changes in free energy. Binding assays can be performed with thetarget fixed to a well, bead or chip or captured by an immobilizedantibody or resolved by capillary electrophoresis. The bound agents maybe detected usually using colorimetric or fluorescence or surfaceplasmon, resonance.

In certain embodiments, high-throughput screening of agents can becarried out to identify agents that affect the biological function ofAMPK. A variety of assay formats will suffice and, in light of thepresent disclosure, those not expressly described herein willnevertheless be comprehended by one of ordinary skill in the art. Asdescribed herein, the test agents of the invention may be created by anycombinatorial chemical method. Alternatively, the subject agents may benaturally occurring biomolecules synthesized in vivo or in vitro. Agentsto be tested for their ability to act as modulators of the biologicalfunction of AMPK can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules; including peptidomimetics), or producedrecombinantly. Test agents contemplated by the present invention includenon-peptidyl organic molecules, peptides, polypeptides, polysaccharides,peptidomimetics, sugars, hormones, and nucleic acid molecules.

The candidate agents of the invention can be provided as single,discrete entities, or provided in libraries of greater complexity, suchas made by combinatorial chemistry. The agents can comprise, forexample, drugs, metabolites, intermediates, cofactors, transition stateanalogs, ions, metals, toxins and natural and synthetic polymers (e.g.,proteins, peptides, nucleic acids, polysaccharides, glycoproteins,hormones, receptors and cell surfaces such as cell walls and cellmembranes. Agents may also comprise alcohols, alkyl halides, amines,amides, esters, aldehydes, ethers and other classes of organic agents.Presentation of candidate agents to the test system can be in either anisolated form or as mixtures of agents, especially in initial screeningsteps. Optionally, the agents may be derivatized with other agents andhave derivatizing groups that facilitate isolation of the agents.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many screening programs which test libraries of agents and naturalextracts, high throughput assays are desirable in order to maximize thenumber of agents surveyed in a given period of time. Assays which areperformed in cell-free systems, such as may be derived with purified orsemi-purified proteins, are often preferred as “primary” screens in thatthey can be generated to permit rapid development and relatively easydetection of an alteration in a molecular target which is mediated by atest agent. Moreover, the effects of cellular toxicity orbioavailability of the test agent can be generally ignored in the invitro system, the assay instead being focused primarily on the effect ofthe agent on the molecular target.

Kits

Also provided herein are kits, e.g., kits for therapeutic purposes orkits for genotyping subjects to identify a reduced MIF expressiongenotype. In one embodiment, a kit comprises at least one containermeans having disposed therein reagents for genotyping a subject for thepresence of a polymorphism associated with reduced MIF expression. Forillustrative purposes, genotyping reagents may include polynucleotideprobes or primers, or solid substrates such as chips or microarrays thatare capable of detecting whether a polymorphism associated with reducedMIF expression is present. For use in a kit, polynucleotides may be anyof a variety of natural and/or synthetic compositions, or chimericmixtures thereof, such as synthetic polynucleotides, restrictionfragments, cDNAs, synthetic peptide nucleic acids (PNAs), and the like.The assay kit and method may also employ labeled polynucleotides toallow ease of identification in the assays. Examples of labels which maybe employed include radiolabels, enzymes, fluorescent compounds,streptavidin, avidin, biotin, magnetic moieties, metal binding moieties,antigen, enzymatic or antibody moieties, and the like. The kit mayoptionally comprise a label and/or instructions for use.

The kit may, optionally, also include DNA sampling means. DNA samplingmeans are well known to one of skill in the art and can include, but notbe limited to substrates, such as filter papers, the AmpliCard™(University of Sheffield, Sheffield, England S10 2JF; Tarlow, J W, etal., J. of Invest. Dematol. 103:387-389 (1994)) and the like; DNApurification reagents such as Nucleon™ kits, lysis buffers, proteinasesolutions and the like; PCR reagents, such as lox reaction buffers,thermostable polymerase, dNTPs, and the like; and allele detection meanssuch as the HinfI restriction enzyme, allele specific oligonucleotides,degenerate oligonucleotide primers for nested PCR from dried blood.Other kit reagents may include enzymes, buffers, small molecules,nucleotides or their analogs, labels (e.g., fluorescent, radioactive,calorimetric, enzymatic or chemical) and/or co-factors as required forthe genotyping assay.

In another embodiment, a kit comprises at least one container meanshaving disposed therein a premeasured dose of one or more MIF agonists.A kit may optionally comprise devices for contacting cells with the MIFagonists and a label and/or instructions for use. Devices includesyringes, dispensers, stents and other devices for introducing a MIFagonist into a subject (e.g., the blood vessel of a subject) or applyingit to the skin of a subject. Kits may also include packaging materialsuch as, but not limited to, ice, dry ice, styrofoam, foam, plastic,cellophane, shrink wrap, bubble wrap, paper, cardboard, starch peanuts,twist ties, metal clips, metal cans, drierite, glass, and rubber (seeproducts available from www.papermart.com, for examples of packagingmaterial).

The practice of the present methods will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2001); DNACloning, Volumes I and II (D. N. Glover ed., 1985); OligonucleotideSynthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195;Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984);Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984);Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide To Molecular Cloning (1984); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells(J. H. Miller and M. P. Calos eds., 1987, Cold Spring HarborLaboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.),Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds., Academic Press, London, 1987); and, Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Wok and C. C. Blackwell, eds., 1986).

EXAMPLES

The following examples are for illustrative purposes and are notintended to be limiting in any way.

Example 1 MIF Stimulation of AMP-Activated Protein Kinase During HypoxiaA. Induction of MIF Expression and AMPK Activation by Hypoxia

Initially, to investigate MIF's role in the stimulation of the AMPKsignaling pathway during hypoxia, we conducted experiments in isolatedrat heart left ventricular papillary muscles.

Briefly, rat heart anterior and posterior left ventricular paillarymuscles (3-5 mg) were equilibrated in oxygenated Dulbecco'sphosphate-buffered saline (PBS) containing 1 mM MgCl₂, 1 mM CaCl₂, 5 mMglucose, and 1% BSA. Unless otherwise noted, 100% oxygen flowedcontinuously through sealed muscle incubation containers while muscleswere kept in an oscillating water bath at 37° C., and experiments beganafter a 15 minute equilibration period in these conditions. Duringincubations designed to examine the effects of hypoxia, muscles weretransferred to incubation containers gassed with N₂ for 30 minutes. Inincubations with rMIF (0-800 ng/ml), anti-MIF blocking antibody (100μg/ml), and/or non-immune rabbit IgG (100 μg/ml; Sigma, USA), thesecompounds were added for 30-120 minutes. At the end of experiments,muscles were freeze clamped and stored in liquid nitrogen until furtheranalysis, unless otherwise specified.

AMPK alpha isoform-specific kinase activity was measured via [³²P]ATP(New England Nuclear, Boston, Mass.) incorporation into the syntheticSAMS peptide after AMPK antibody immuno0isolation (α1 or α2; Santa CruzBiotechnology, Santa Cruz, Calif.) coupled to protein G/A sepharosebeads.

Hypoxia stimulated MIF release into the incubation media, as measured byELISA (FIG. 1B), and led to AMPK alpha subunit Thr172 phosphorylationand increased activity of both AMPK alpha catalytic isoforms, α1 and α2(FIG. 1A). Thirty minutes of hypoxia caused a 2-fold increase in MIFproduction. Pre-treatment of the heart muscles with anti-MIFimmuno-neutralizing antibody reduced the hypoxic activation of AMPK by67% (FIG. 1C). In contrast, control experiments showed no inhibition ofAMPK activation by pre-incubation with non-immune rabbit IgG. Hypoxicactivation of glucose transport was also inhibited by the addition ofanti-MIF, while non-immune IgG had no inhibitory effect (FIG. 1D).

B. Activation of AMPK by Recombinant Human MIF

To further support MIF's role in modulating the AMPK pathway, we addedrecombinant human MIF (rMIF) to intact normoxic heart muscles. Papillarymuscles were isolated as above. Following pre-incubation with rMIF,2-deoxy-[1-³H]glucose (1 μCi/ml) was added during the final 60 minutesof the experiment to measure the rates of glucose transport andphosphorylation. To assess the ability of MIF immuno-neutralization toblock hypoxia-stimulated glucose uptake, papillary muscles werepre-incubated with anti-MIF immuno-neutralizing antibody or rabbit IgGfollowed by incubation in hypoxic media containing2-deoxy-[1-³H]glucose. To correct for the muscle extracellular space andextracellular deoxyglucose, [U-¹⁴C] mannitol (0.1 μCi/ml) was alsoadded. After incubations, muscles were washed in ice-cold PBS, blotteddry, weighed, solubilized in Soluene-350 (Packard Instrument, Meriden,Conn.) and counted by liquid scintillation.

MIF caused a time- and dose-dependent increase in AMPK. Thr172phosphorylation, with maximal AMPK activation (1.5-2.0 fold increase)seen with 400 ng/ml rMIF incubated for 60 minutes (FIGS. 1E and 1F).Likewise, parallel downstream AMPK pathway activation was evident withan increase in glucose uptake after the addition of rMIF to normoxicheart muscles (FIG. 1G).

Glucose uptake is primarily mediated by the glucose transporter, GLUT4,and AMPK-stimulation of glucose transport is due to the translocation ofGLUT4 vesicles to the cell surface, where GLUT4 is physiologicallyactive (Sun et al., Circulation 89, 793 (1994); Young et al.,Circulation 95, 415 (1997)). Thus, we assessed the cell-surface GLUT4content of heart muscles after incubation with rMIF, utilizing acell-membrane impermeant biotinylated glucose transporter photolabel(bio-LC-ATB-BGPA).

Incubation with rMIF increased cell-surface GLUT4 in the heart muscles(FIG. 1H), providing a mechanism for increased glucose uptake during MIFincubation. To measure cell surface GLUT4, papillary muscles were rinsedin ice-cold glucose-free Krebs-Henseleit buffer (KHB) and incubated incold KIM containing 400 μmol/L bio-LC-ATB-BGPA(4,4-O-[2-[2-[2-[2-[2-[6-(biotinylamino)hexanolyl]-amino]ethoxy]ethoxy]-ethoxy]-4-1-azi-2,2,2-trifluoroethyl)benzoyl]amino-1,3-prpanediyl]bis-D-glucose).Following cross-linking of bio-LC-ATB-BGPA to cell-surface glocosetransporters by UV irradiation, cell surfact GLUT4 was isolated onstreptavidin-agarose (Pierce Biotechnology, Rockford, Ill.), identifiedby immunoblotting and quantified by densitometry.

Thus, these data taken together provide evidence that MIF is released inheart muscle in response to hypoxia, leading to activation of AMPK,stimulation of GLUT4 translocation and glucose uptake.

Example 2 MIF Stimulates AMP-Activated Protein Kinase in Ischemia

We next examined the role of MIF in AMPK signaling in ischemia utilizingisolated mouse heart perfused with a crystalloid buffer to eliminate thepotential contribution of MIF from circulating immune cells or monocytesduring ischemia.

A. Determination of MIF Expression in Ischemia

Immunoblotting of whole-heart lysates showed high-level MIF proteinexpression in the heart, and immunohistochemistry of heart sectionsdemonstrated cardiomyocyte-predominant MIF staining in hearts fromwild-type mice following control perfusions (FIG. 2A). Male MIF −/− micewere compared to age-matched wild-type BALB/c controls. Mouse heartswere retrogradely perfused in the Langendorff mode with modified KHBbuffer containing 7 mmol/L glucose, 1.0 mmol/L oleate, 1% BSA, and a lowfasting concentration of insulin (10 μU/mL). Hearts were perfused for 30minutes at a flow of 4 mL/min, followed by either: 20 minutes of globalischemia, 20 minutes of global ischemia followed by 30 minutes ofreperfusion, or control perfusion of the appropriate duration atcontinued baseline flow. Cardiac function was measured by a fluid-filledballoon inserted into the left ventricular cavity, connected to a Millartransducer (Millar Instruments, Houston, Tex.) and acquired via anADInstruments PowerLab system with Chart v.5.2.2 software(ADInstruments, Colorado Springs, Colo.). Hearts were freeze-clamped inliquid nitrogen at the end of the perfusions unless otherwise noted. Forimmunohistochemistry, hearts were fixed with 4% formalin afterperfusions and infused via the aortic cannula followed by immersion,paraffin embedded, and 3 μm sections cut with a tissue microtome.Sections were immunostained using a DakoCytomation EnVision+ System-HRP(DAB) (DakoCytomation, Carpinteria, Calif.) at room temperature afterd-parafinization with xylene and ethanol. Endogenous peroxidases wereblocked using Dako Peroxidase Block, followed by blocking ofnon-specific antibody binding sites with 10% bovine serum albuminPrimary antibody incubations were preliminarily performed at multipledilutions to establish optimum signal:background recovery, leading touse of a final primary antibody dilution of 1:1000 incubated for 4hours. Secondary antibody incubation with labeled polymer-HRPanti-rabbit antibody was followed by developing with DAB andcounterstaining with hematoxylin. Negative controls for each heartincluded use of non-specific rabbit IgG (Santa Cruz, Santa Cruz, Calif.)and secondary antibody alone. Specificity of the anti-MIF wasdemonstrated by lack of staining of sections from MIF −/− tissues. Allsections were processed simultaneously, developed for identical periods,and analyzed by light microscopy after dehydration with ethanol andxylene and mounting under coverslips.

Immunoblots were performed according to standard methods known in theart. See, for example, Li et al., Am. J. Physiol Endocrinol Metab 287,E384 (2004) and Baron et al., Circ Res (Jan. 13, 2005). Heart homogenateproteins were combined with Laemmli sample buffer prior to resolution bySDS-PAGE, transferred onto polyvinylidene difluoride membranes,immunoblotted, detected with enhanced chemiluminescence, and quantifiedby densitometry.

During cardiac perfusion in the Langendorff mode, MIF was released fromhearts into the coronary venous effluent, but heart MIF release wasincreased 2-fold during reperfusion after ischemic (3.2±0.43 vs. 1.5±0.3ng/min/g, P=1.01) which correlated with a significant decrease in hearthomogenate MIF levels (0.96±0.15 vs. 0.43±0.010 relative units, P=1.03)(FIG. 2B). Increased MIF release from the heart during reperfusion afterthis brief period of ischemia was not associated with evidence ofmyocardial necrosis, based the results of vital staining and creatinekinase measurement on the coronary effluent.

B. AMPK Signaling in the Presence and Absence of MIF

To determine the role of MIF in ischemic cardiac AMPK activation, weexamined AMPK signaling in hearts from transgenic MIF−/− mice comparedto wild-type (WT) controls. The generation of germline MIF−/− mice hasbeen previously described (Bozza et al., J Exp Med 189, 341 (1999)). NoMIF was detected by immunohistochemistry or immunoblotting in MIF−/−hearts (FIG. 2A). The cardiac phenotype of the MIF−/− mice wascharacterized. Normal cardiac chamber sizes and function were found byechocardiography. There were no evident histological abnormalities, andnormal heart weight:body weight ratio and normal expression of totalAMPK, GLUT1, and GLUT4 were found (FIG. 5). MIF−/− and WT hearts wereperfused with a mixed-substrate buffer in the Langendorff mode andsubjected to 15 minutes of global, no-flow ischemia.

Following ischemia, AMPK phosphorylation and activity were decreased inthe MIF−/− hearts (FIG. 3A). This defect in AMPK signaling translated toa defect in glucose uptake following ischemia in the MIF−/− hearts (FIG.3B). Rates of glucose uptake were similar in WT and MIF−/− hearts duringcontrol perfusions. During post-ischemic reperfusion, however,stimulation of glucose uptake was significantly blunted in MIF−/−compared to WT hearts (P=0.04). Consistent with impaired glucose uptakeduring ischemic reperfusion, MIF−/− hearts also had diminished glycogensynthesis compared to WT hearts, despite similar calculated amount ofischemic glycogen breakdown (FIG. 6).

Defective AMPK signaling in the MIF−/− hearts impaired their ischemictolerance. Baseline cardiac function was similar in WT and MIF−/− heartsperfused with mixed substrate buffer (FIG. 3C). However, when reperfusedfollowing ischemia, MIF−/− hearts had impaired recovery of cardiacfunction compared to WT controls, as evidenced by reduced leftventricular rate-pressure product (FIG. 3C), as well as depressed leftventricular developed pressure and contractility (+dp/dt) (FIG. 7). Onaverage, MIF−/− hearts recovered 51±6.1% of their pre-ischemic cardiacfunction, while WT hearts recovered 81±8.7% (P=0.03). These resultssupport the contention that MIF activation of AMPK duringischemia/reperfusion promotes early adaptive metabolic responses in theheart, while also elucidating a new physiologic action for MIF in theheart.

Example 3 Effect of Polymorphisms in the Human MIF Promoter on MIFSecretion and AMPK Activation

We also examined the effect of polymorphisms in the human MIF promoteron functional differences in MIF secretion and cellular AMPK activation.Early passage human dermal fibroblasts were subjected to hypoxiatreatment and the degree of AMPK activation was assessed in relation totheir MIF promoter polymorphism alleles.

To isolate human fibroblasts, human foreskin samples were obtained fromthe Yale Human Cell Resource Center (Department of Dermatology) inaccordance with the Yale Human Investigations Committee. Fibroblastswere cultured in DMEM supplemented with 10% fetal bovine serum (FBS),penicillin (100 IU/ml) and streptomycin (100 μg/ml) at 37° C. Cells werestudied at 80% confluence, and on the day prior to experiments, culturemedia was changed to DMEM with 1% PBS. Early passage fibroblasts wereeither subjected to 9 hours of hypoxia by incubation in an air-tightchamber gassed with 95% nitrogen/5% CO₂ or control incubation in roomair.

Three of seven specimens carried two 5-CATT alleles (“515” genotype)with the remainder baying at least one 6, 7, or 8-CATT repeat alleles(“non-5/5” genotype). Fibroblasts with the non-5/5 genotype hadsignificantly greater MIF release into the culture media both basally(P=0.03), as well as after hypoxia (P=0.05), compared to 5/5 genotypecells (FIG. 4A). We found that greater MIF release from the non-5/5genotype fibroblasts was associated with significantly increased hypoxicAMPK phosphorylation compared to fibroblasts with the 5/5 MIF promotergenotype (FIG. 4B).

To confirm that relative MIF deficiency was responsible for impairedhypoxic AMPK activation in the low-expressing 5/5 genotype fibroblasts,we investigated the effect of adding human rMIF (10 ng/ml) to theculture media during hypoxic incubation. The addition of rMIF “rescued”hypoxic AMPK signaling in the 5/5 genotype fibroblasts, increasinghypoxic AMPK phosphorylation to levels that were equivalent to thenon-5/5 fibroblasts, but had no additional effect on AMPKphosphorylation in hypoxic non-5/5 fibroblasts (FIG. 4B). These resultsdemonstrate differential MIF release governed by a common polymorphismin the human MIF gene promoter that has consequences in stresssignaling, specifically modulating hypoxic AMPK signaling. Takentogether with the above results implicating MIF in the activation ofAMPK in ischemia, these data indicate that common polymorphisms in theMIF promoter gene may be used as indicators of susceptibility toischemic injury, particularly in patients with coronary artery disease.

Statistical data were expressed as means±standard deviation. A value ofP<0.05 was considered significant. Significance was tested by Student2-tail t tests or 2-way repeated measures ANOVA with Bonferronicorrection for multiple comparisons when appropriate, using GraphPadPrism 4.02.

A rabbit polyclonal antibody raised to pure rMIF was used forimmunohistochemistry, and a rabbit anti-rat MIF antibody (Torry PinesBiolabs, Houston, Tex.) was used for immunoblotting. Rabbit monoclonalanti-AMPK p-Thr¹⁷² (Cell Signaling, Beverly, Mass.), rabbit anti-AMPKalpha (Cell Signaling, Beverly, Mass.), rabbit anti-GLUT4, andHRP-conjugated streptavidin (Pierce, Rockford, Ill.) were used forrespective immunoblosts as described.

Animals were housed in accordance with guidelines from the AmericanAssociation for Laboratory Animal Care. All procedures were approved bythe Yale University Animal Care and Use Committee. All animals werehoused in a 12-hour light/dark cycle and allowed standard chow and waterad libitum. Male Sprague-Dawley rats weighing 250-275 g were purchasedfrom Charles River Laboratories. Wild-type male BALB/c mice (age 10-16weeks) were purchased from Charles River Laboratories and compared toMIF −/− mice (generation N8) bred at the Yale Animal Resources Center onthe BALB/c genetic background.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The full scope of the inventionshould be determined by reference to the claims, along with their fullscope of equivalents, and the specification, along with such variations.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference. In vase ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) (www.tigr.org) and/or theNational Center for Biotechnology Information (NCBI)(www.ncbi.nlm.nih.gov).

What is claimed is:
 1. A method of identifying a subject at risk ofdeveloping a condition in which increased AMPK activity is desirable,comprising genotyping the subject for the presence of a polymorphismassociated with decreased MIF expression, wherein a subject having apolymorphism associated with decreased MIF expression is at an increasedrisk of developing a condition in which increased AMPK activity isdesirable.
 2. The method of claim 1, wherein the condition in whichincreased AMPK activity is desirable is a condition selected from thegroup consisting of hypoxia, ischemia, and type 2 diabetes.
 3. Themethod of claim 2, wherein the ischemia is caused by myocardialinfarction, coronary revascularization, stroke, vascular occlusion,organ transplant surgery, vascular surgery.
 4. A method of predictingthe severity of a condition in which increased AMPK activity isdesirable in a subject, comprising genotyping the subject for thepresence of a polymorphism associated with decreased MIF expression,wherein a subject having a polymorphism associated with decreased MIFexpression is at an increased risk of developing a more severe conditionin which increased AMPK activity is desirable.
 5. The method of claim 4,wherein the condition in which increased AMPK activity is desirable is acondition selected from the group consisting of: hypoxia, ischemia, andtype 2 diabetes.
 6. The method of claim 5, wherein the ischemia iscaused by myocardial infarction, coronary revascularization, stroke,vascular occlusion, organ transplant surgery, vascular surgery.
 7. Amethod of predicting whether a subject is susceptible to a condition inwhich increased AMPK activity is desirable, comprising genotyping asubject for the presence of a polymorphism associated with decreased MIFexpression, wherein a subject having a polymorphism associated withdecreased MIF expression is more susceptible to the condition.
 8. Themethod of claim 7, wherein the condition in which increased AMPKactivity is desirable is a condition selected from the group consistingof hypoxia, ischemia, and type 2 diabetes.
 9. The method of claim 8,wherein the ischemia is caused by myocardial infarction, coronaryrevascularization, stroke, vascular occlusion, organ transplant surgery,vascular surgery.
 10. The method of claim 1, wherein the polymorphismassociated with decreased MIF expression is selected from the groupconsisting of: the presence of five CATT repeats in the −794 region ofone or both alleles of the MIF gene, the presence of fewer than fiveCATT repeats in the −794 region of one or both alleles of the MIF gene,or the presence of guanine at position −173 of one or both alleles ofthe MIF gene.
 11. The method of claim 1, wherein a subject having apolymorphism associated with decreased MIF expression is a subjecthaving a polymorphism selected from the group consisting of: thepresence of five CATT repeats in the −794 region of one or both allelesof the MIF gene, the presence of fewer than five CATT repeats in the−794 region of one or both alleles of the MIF gene, or the presence ofguanine at position −173 of one or both alleles of the MIF gene.
 12. Themethod of claim 1, wherein genotyping the subject for the presence of apolymorphism associated with decreased MIF expression comprises: (a)contacting a sample obtained from the subject with a polynucleotideprobe that hybridizes specifically to a sequence comprising apolymorphism associated with decreased MIF expression; and (b)determining whether hybridization occurs, wherein hybridizationindicates whether the subject comprises a polymorphism associated withdecreased MIF expression, thereby genotyping the subject for thepresence of a polymorphism associated with decreased MIF expression. 13.The method of claim 12, wherein the method further comprises: (c)contacting the sample with a control polynucleotide probe, wherein thecontrol polynucleotide probe does not hybridize specifically to asequence comprising a polymorphism associated with decreased MIFexpression, and wherein hybridization of the polynucleotide probe butnot the control polynucleotide probe indicates the presence of a MIFpolymorphism associated with decreased MIF expression.
 14. The method ofclaim 1, wherein genotyping the subject for the presence of apolymorphism associated with decreased MIF expression comprises: (a)contacting a sample obtained from the subject with a pair ofamplification primers, wherein said primers are capable of amplifying aportion of the MIF promoter comprising a polymorphism associated withdecreased MIF expression; (b) amplifying DNA in the sample, therebyproducing amplified DNA; (c) determining whether the amplified DNAcomprises a polymorphism associated with decreased MIF expression,thereby genotyping the subject for the presence of a polymorphismassociated with decreased MIF expression.
 15. The method of claim 14,wherein the determining step comprises sequencing the amplified DNA. 16.A method of increasing phosphorylation of threonine at position 172 ofthe AMPK protein in a cell, comprising administering a MIF agonist to asubject in need thereof.
 17. A method of increasing AMPK-mediated GLUT4activation in a cell, comprising administering a MIF agonist to asubject in need thereof.
 18. A method of increasing uptake ofAMPK-mediated glucose into a cell, comprising administering a MIFagonist to a subject in need thereof.
 19. A method of increasingAMPK-mediated glycogen synthesis in a cell, comprising administering aMIF agonist to a subject in need thereof.
 20. A method of stimulatingAMPK-mediated PFK-2 activity in a cell, comprising administering a MIFagonist to a subject in need thereof.
 21. A method of increasingAMPK-mediated glycolysis in a cell, comprising administering a MIFagonist to a subject in need thereof.
 22. A composition comprising oneor more MIF agonists and at least one AMPK agonist.
 23. A compositioncomprising one or more MIF agonists and at least one additionaltherapeutic agent.
 24. The composition of claim 23, wherein said atleast one additional therapeutic agent is a compound for treating asubject having, or at risk of developing, ischemia, organ transplantsurgery, and type 2 diabetes
 25. The composition of claims 22, whereinthe MIF agonist is selected from the group consisting of: a MIFpolypeptide, a CD74 agonist, a CD44 agonist, a bivalent antibody thatincreases the interaction between MIF, CD74 and CD44, a bivalentantibody that increases the interaction between MIF and CD74, and abivalent antibody that increases the interaction between CD74 and CD44,and a polynucleotide or cDNA molecule that encodes a MIF polypeptide, aCD74 agonist, a CD44 agonist, a bivalent antibody that increases theinteraction between MIF, CD74 and CD44, a bivalent antibody thatincreases the interaction between MIF and CD74, a bivalent antibody thatincreases the interaction between CD74 and CD44, and any combinationthereof.